Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.
Enzymes catalyze a vast range of reactions. Their catalytic performances, mechanisms, global folds, and active-site architectures are also highly diverse, suggesting that enzymes are shaped by an entire range of physiological demands and evolutionary constraints, as well as by chemical and physicochemical constraints. We have attempted to identify signatures of these shaping demands and constraints. To this end, we describe a bird's-eye view of the enzyme space from two angles: evolution and chemistry. We examine various chemical reaction parameters that may have shaped the catalytic performances and active-site architectures of enzymes. We test and weigh these considerations against physiological and evolutionary factors. Although the catalytic properties of the "average" enzyme correlate with cellular metabolic demands and enzyme expression levels, at the level of individual enzymes, a multitude of physiological demands and constraints, combined with the coincidental nature of evolutionary processes, result in a complex picture. Indeed, neither reaction type (a chemical constraint) nor evolutionary origin alone can explain enzyme rates. Nevertheless, chemical constraints are apparent in the convergence of active site architectures in independently evolved enzymes, although significant variations within an architecture are common.
Marine organisms release dimethylsulfide (DMS) via cleavage of dimethylsulfoniopropionate (DMSP). Different genes encoding proteins with DMSP lyase activity are known, yet these exhibit highly variable levels of activity. Most assigned bacterial DMSP lyases, including DddK, DddL, DddQ, DddW, and DddY, appear to belong to one, cupin-like superfamily. Here, we attempted to define and map this superfamily dubbed cupin-DLL (DMSP lyases and lyase-like). To this end, we have pursued the characterization of various recombinant DMSP lyases belonging to this superfamily of metalloenzymes, and especially of DddY and DddL that seem to be the most active DMSP lyases in this superfamily. We identified two conserved sequence motifs that characterize this superfamily. These motifs include the metal-ligating residues that are absolutely essential and other residues including an active site tyrosine that seems to play a relatively minor role in DMSP lysis. We also identified a transition metal chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN), that selectively inhibits all known members of the cupin-DLL superfamily that exhibit DMSP lyase activity. A phylogenetic analysis indicated that the known DMSP lyase families are sporadically distributed suggesting that DMSP lyases evolved within this superfamily multiple times. However, unusually low specific DMSP lyase activity and genome context analysis suggest that DMSP lyase is not the native function of most cupin-DLL families. Indeed, a systematic profiling of substrate selectivity with a series of DMSP analogues indicated that some members, most distinctly DddY and DddL, are bona fide DMSP lyases, while others, foremost DddQ, may only exhibit promiscuous DMSP lyase activity.
Rational design and directed evolution have proved to be successful approaches to increase catalytic efficiencies of both natural and artificial enzymes. Protein dynamics is recognized as important, but due to the inherent flexibility of biological macromolecules it is often difficult to distinguish which conformational changes are directly related to function. Here, we use directed evolution on an impaired mutant of the proline isomerase CypA and identify two second-shell mutations that partially restore its catalytic activity. We show both kinetically, using NMR spectroscopy, and structurally, by room-temperature X-ray crystallography, how local perturbations propagate through a large allosteric network to facilitate conformational dynamics. The increased catalysis selected for in the evolutionary screen is correlated with an accelerated interconversion between the two catalytically essential conformational substates, which are both captured in the high-resolution X-ray ensembles. Our data provide a glimpse of an evolutionary trajectory and show how subtle changes can fine-tune enzyme function.
The practical need for highly efficient enzymes presents new challenges in enzyme engineering, in particular, the need to improve catalytic turnover (k(cat)) or efficiency (k(cat)/K-M) by several orders of magnitude. However, optimizing catalysis demands navigation through complex and rugged fitness landscapes, with optimization trajectories often leading to strong diminishing returns and dead-ends. When no further improvements are observed in library screens or selections, it remains unclear whether the maximal catalytic efficiency of the enzyme (the catalytic 'fitness peak') has been reached; or perhaps, an alternative combination of mutations exists that could yield additional improvements. Here, we discuss fundamental aspects of the process of catalytic optimization, and offer practical solutions with respect to overcoming optimization plateaus.
The linkage between regulatory elements of transcription, such as promoters, and their protein products is central to gene function. Promoter-protein coevolution is therefore expected, but rarely observed, and the manner by which these two regulatory levels are linked remains largely unknown. We study glutamate dehydrogenase-a hub of carbon and nitrogen metabolism. In Bacillus subtilis, two paralogues exist: GudB is constitutively transcribed whereas RocG is tightly regulated. In their active, oligomeric states, both enzymes show similar enzymatic rates. However, swaps of enzymes and promoters cause severe fitness losses, thus indicating promoter-enzyme coevolution. Characterization of the proteins shows that, compared to RocG, GudB's enzymatic activity is highly dependent on glutamate and pH. Promoter-enzyme swaps therefore result in excessive glutamate degradation when expressing a constitutive enzyme under a constitutive promoter, or insufficient activity when both the enzyme and its promoter are tightly regulated. Coevolution of transcriptional and enzymatic regulation therefore underlies paralogue-specific spatio-temporal control, especially under diverse growth conditions.
Under stress, metabolism is changing: specific up- or down-regulation of proteins and metabolites occurs as well as side effects. Distinguishing specific stress-signaling metabolites (alarmones) from side products (damage metabolites) is not trivial. One example is diadenosine tetraphosphate (Ap4A) - a side product of aminoacyl-tRNA synthetases found in all domains of life. The earliest observations suggested that Ap4A serves as an alarmone for heat stress in Escherichia coli. However, despite 50 years of research, the signaling mechanisms associated with Ap4A remain unknown. We defined a set of criteria for distinguishing alarmones from damage metabolites to systematically classify Ap4A. In a nutshell, no indications for a signaling cascade that is triggered by Ap4A were found; rather, we found that Ap4A is efficiently removed in a constitutive, nonregulated manner. Several fold perturbations in Ap4A concentrations have no effect, yet accumulation at very high levels is toxic due to disturbance of zinc homeostasis, and also because Ap4A's structural overlap with ATP can result in spurious binding and inactivation of ATP-binding proteins. Overall, Ap4A met all criteria for a damage metabolite. While we do not exclude any role in signaling, our results indicate that the damage metabolite option should be considered as the null hypothesis when examining Ap4A and other metabolites whose levels change upon stress.
Determining the properties of proteins prior to purification saves time and labor. Here, we demonstrate a native mass spectrometry approach for rapid characterization of overexpressed proteins directly in crude cell lysates. The method provides immediate information on the identity, solubility, oligomeric state, overall structure, and stability, as well as ligand binding, without the need for purification.
Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (k(cat)/K-M) of 5 x 10(6)M(-1) min(-1). However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of > 10(7) M-1 min(-1). Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a k(cat)/K-M >= 5 x 10(7)M(-1) min(-1) and Russian VX (RVX) with a k(cat)/K-M >= 10(7) M-1 min(-1). These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by > 5000-fold with VX and by > 17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at k(cat)/K-M > 5 x 10(7) M-1 min(-1)) and can thus serve as candidates for broadspectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.
S-Adenosylmethionine (SAM) is an essential methylation cofactor. The origins of SAM methylation are complex, seemingly demanding the simultaneous emergence of an enzyme that makes SAM and enzyme(s) that utilize it. We report that both ATP and adenosine spontaneously react with methionine to yield SAM, thus suggesting that SAM could have emerged by chance. SAM methylation thus exemplifies how metabolites and pathways can co-emerge through the gradual recruitment of individual enzymes in reverse order.
Atmospheric dimethylsulfide (DMS) is massively produced in the oceans by bacteria, algae, and corals. To enable identification of DMS sources, we developed a potent mechanism-based inhibitor of the algal Alma dimethylsulfoniopropionate lyase family that does not inhibit known bacterial lyases. Its application to coral holobiont indicates that DMS originates from Alma lyase(s). This biochemical profiling may complement meta-genomics and transcriptomics to provide better understanding of the marine sulfur Cycle.
Organophosphate (OP) based pesticides are highly toxic compounds that are still widely used in agriculture around the world. According to World Health Organization (WHO) data, it is estimated that between 250,000 and 370,000 deaths occur yearly around the globe as a result of acute intoxications by pesticides. Currently available antidotal drug treatments of severe OP intoxications are symptomatic, do not reduce the level of intoxicating OP in the body and have limited ability to prevent long-term brain damage. Pesticide poisonings present a special therapeutic challenge since in many cases, such as with parathion, their toxicity stems from their metabolites that inhibit the essential enzyme acetylcholinesterase. Our goal is to develop a new treatment strategy for parathion intoxication by combining a catalytic bioscavenger that rapidly degrades the intoxicating parathion-metabolite (paraoxon) in the blood, with a glutamate bioscavenger that reduces the elevated concentration of extracellular glutamate in the brain following OP intoxication. We report on the development of a novel catalytic bioscavenger by directed evolution of serum paraoxonase 1 (PON1) that effectively detoxifies paraoxon in-vivo. We also report preliminary results regarding the utilization of this PON1 variant together with a recombinant human enzyme glutamate oxaloacetate transaminase 1 (rGOT1), suggesting that a dual PON-GOT treatment may increase survival and recovery from parathion and paraoxon intoxications. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
The nearly 200,000 fatalities following exposure to organophosphorus (OP) pesticides each year and the omnipresent danger of a terroristic attack with OP nerve agents emphasize the demand for the development of effective OP antidotes. Standard treatments for intoxicated patients with a combination of atropine and an oxime are limited in their efficacy. Thus, research focuses on developing catalytic bioscavengers as an alternative approach using OP-hydrolyzing enzymes such as Brevundimonas diminuta phosphotriesterase (PTE). Recently, a PTE mutant dubbed C23 was engineered, exhibiting reversed stereoselectivity and high catalytic efficiency (k (cat)/K (M)) for the hydrolysis of the toxic enantiomers of VX, CVX, and VR. Additionally, C23's ability to prevent systemic toxicity of VX using a low protein dose has been shown in vivo. In this study, the catalytic efficiencies of V-agent hydrolysis by two newly selected PTE variants were determined. Moreover, in order to establish trends in sequence-activity relationships along the pathway of PTE's laboratory evolution, we examined k (cat)/K (M) values of several variants with a number of V-type and G-type nerve agents as well as with different OP pesticides. Although none of the new PTE variants exhibited k (cat)/K (M) values > 10(7) M-1 min(-1) with V-type nerve agents, which is required for effective prophylaxis, they were improved with VR relative to previously evolved variants. The new variants detoxify a broad spectrum of OPs and provide insight into OP hydrolysis and sequence-activity relationships.
Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown.
Ancestral reconstruction provides instrumental insights regarding the biochemical and biophysical characteristics of past proteins. A striking observation relates to the remarkably high thermostability of reconstructed ancestors. The latter has been linked to high environmental temperatures in the Precambrian era, the era relating to most reconstructed proteins. We found that inferred ancestors of the serum paraoxonase (PON) enzyme family, including the mammalian ancestor, exhibit dramatically increased thermostabilities compared with the extant, human enzyme (up to 30 degrees C higher melting temperature). However, the environmental temperature at the time of emergence of mammals is presumed to be similar to the present one. Additionally, the mammalian PON ancestor has superior folding properties (kinetic stability)-unlike the extant mammalian PONs, it expresses in E. coli in a soluble and functional form, and at a high yield. We discuss two potential origins of this unexpectedly high stability. First, ancestral stability may be overestimated by a "consensus effect," whereby replacing amino acids that are rare in contemporary sequences with the amino acid most common in the family increases protein stability. Comparison to other reconstructed ancestors indicates that the consensus effect may bias some but not all reconstructions. Second, we note that high stability may relate to factors other than high environmental temperature such as oxidative stress or high radiation levels. Foremost, intrinsic factors such as high rates of genetic mutations and/or of transcriptional and translational errors, and less efficient protein quality control systems, may underlie the high kinetic and thermodynamic stability of past proteins.
The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (similar to 2LD(50)). C23AL showed a C-max of 0.63 mu mol L (1) after i.o. and i.v. administration of 2 mg kg (1) providing a stable plasma profile up to 180 min experimental duration with 0.41 and 0.37 mu mol L (1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning. (C) 2016 Elsevier Irela
The last decade has seen a growing number of experiments aimed at systematically mapping the effects of mutations in different proteins, and of attempting to correlate their biophysical and biochemical effects with organismal fitness. While insightful, systematic laboratory measurements of fitness effects present challenges and difficulties. Here, we discuss the limitations associated with such measurements, and in particular the challenge of correlating the effects of mutations at the single protein level ("protein fitness") with their effects on organismal fitness. A variety of experimental setups are used, with some measuring the direct effects on protein function and others monitoring the growth rate of a model organism carrying the protein mutants. The manners by which fitness effects are calculated and presented also vary, and the conclusions, including the derived distributions of fitness effects of mutations, vary accordingly. The comparison of the effects of mutations in the laboratory to the natural protein diversity, namely to amino acid changes that have fixed in the course of millions of years of evolution, is also debatable. The results of laboratory experiments may, therefore, be less relevant to understanding long-term inter-species variations yet insightful with regard to short-term polymorphism, for example, in the study of the effects of human SNPs.
Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase ( hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at similar to 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
Derived from the yeast whole-genome duplication, Saccharomyces cerevisiae GAL1 and GAL3 encode the catabolic enzyme galactokinase (Gal1) and its transcriptional coinducer (Gal3), whereas the ancestral, preduplicated GAL1 gene performed both functions. Previous studies indicated that divergence was primarily driven by changes in upstream promoter elements, and changes in GAL3's coding region are assumed to be the result of drift. We show that replacement of GAL3's open-reading-frame with GAL1's results in an extended lag phase upon switching to growth on galactose with up to 2.5-fold differences in the initial cell masses. Accordingly, the binding affinity of Gal3 to Gal80 was found to be greater than 10-folds higher than that of Gal1, with both a higher association rate (k(a)) and lower dissociation (k(d)) rate. Thus, while changes in the noncoding, regulatory regions were the initial driving force for GAL3's subfunctionalization as a coinducer, adaptive changes in the protein sequence seem to have followed.
Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of b-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller- like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandemduplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.
Ancestral reconstruction is a powerful tool for studying protein evolution as well as for protein design and engineering. However, in many positions alternative predictions with relatively high marginal probabilities exist, and thus the prediction comprises an ensemble of near-ancestor sequences that relate to the historical ancestor. The ancestral phenotype should therefore be explored for the entire ensemble, rather than for the sequence comprising the most probable amino acid at all positions [the most probable ancestor (mpa)]. To this end, we constructed libraries that sample ensembles of near-ancestor sequences. Specifically, we identified positions where alternatively predicted amino acids are likely to affect the ancestor's structure and/or function. Using the serum paraoxonases (PONs) enzyme family as a test case, we constructed libraries that combinatorially sample these alternatives. We next characterized these libraries, reflecting the vertebrate and mammalian PON ancestors. We found that the mpa of vertebrate PONs represented only one out of many different enzymatic phenotypes displayed by its ensemble. The mammalian ancestral library, however, exhibited a homogeneous phenotype that was well represented by the mpa. Our library design strategy that samples near-ancestor ensembles at potentially critical positions therefore provides a systematic way of examining the robustness of inferred ancestral phenotypes.
Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids beta-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to + 1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.
Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.
Copyright 2015 Elsevier Ltd. All rights reserved.Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis-a complete reversion of mutational effects, particularly in protein evolution-yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 beta-lactamase- an enzyme that endows antibiotics resistance. Separated by 10A, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.
Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design. (C) 2015 Elsevier Ltd. All rights reserved.
To carry out their activities, biological macromolecules balance different physical traits, such as stability, interaction affinity, and selectivity. How such often opposing traits are encoded in a macromolecular system is critical to our understanding of evolutionary processes and ability to design new molecules with desired functions. We present a framework for constraining design simulations to balance different physical characteristics. Each trait is represented by the equilibrium fractional occupancy of the desired state relative to its alternatives, ranging from none to full occupancy, and the different traits are combined using Boolean operators to effect a "fuzzy"-logic language for encoding any combination of traits. In another paper, we presented a new combinatorial backbone design algorithm AbDesign where the fuzzy-logic framework was used to optimize protein backbones and sequences for both stability and binding affinity in antibody-design simulation. We now extend this framework and find that fuzzy-logic design simulations reproduce sequence and structure design principles seen in nature to underlie exquisite specificity on the one hand and multispecificity on the other hand. The fuzzy-logic language is broadly applicable and could help define the space of tolerated and beneficial mutations in natural biomolecular systems and design artificial molecules that encode complex characteristics. (C) 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.
The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(-) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (k(cat)/K-M = 5 x 10(6) M-1 min(-1)). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5 mg kg(-1) i.v.) 5 min after s.c. challenge with VX (similar to 2LD(50)) prevented systemic toxicity. A lower C23 dose (2 mg kg(-1)) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15 min post VX) with 5 mg kg(-1) C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(-)-VX enantiomer that correlates with the C23 blood levels and its k(cat)/K-M value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single postexposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication. (C) 2014 Elsevier Ireland Ltd. All rights
Dimethyl sulfide (DMS) is produced in oceans in vast amounts (>10(7) tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddD's native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.
I discuss some physico-chemical and evolutionary aspects of enzyme accuracy (selectivity, specificity) end speed (turnover rate, processivity). Accuracy can be a beneficial side-product of active-sites being refined to proficiently convert a given substrate into one product. However, exclusion of undesirable, non-cognate substrates is also an explicitly evolved trait that may come with a cost. I define two schematic mechanisms. Ground-state discrimination applies to enzymes where selectivity is achieved primarily at the level of substrate binding. Exemplified by DNA methyltransferases and the ribosome, ground-state discrimination imposes strong accuracy-rate tradeoffs. Alternatively, transition-state discrimination, applies to relatively small substrates where substrate binding and chemistry are efficiently coupled, and evokes weaker tradeoffs. Overall, the mechanistic, structural and evolutionary basis of enzymatic accuracy-rate tradeoffs merits deeper understanding.
The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (similar to 2LD(50))-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.
Assignment of protein folds to functions indicates that >60% of folds carry out one or two enzymatic functions, while few folds, for example, the TIM-barrel and Rossmann folds, exhibit hundreds. Are there structural features that make a fold amenable to functional innovation (innovability)? Do these features relate to robustness - the ability to readily accumulate sequence changes? We discuss several hypotheses regarding the relationship between the architecture of a protein and its evolutionary potential. We describe how, in a seemingly paradoxical manner, opposite properties, such as high stability and rigidity versus conformational plasticity and structural order versus disorder, promote robustness and/or innovability. We hypothesize that polarity - differentiation and low connectivity between a protein's scaffold and its active-site - is a key prerequisite for innovability.
Organismal adaptation to extreme temperatures yields enzymes with distinct configurational stabilities, including thermophilic and psychrophilic enzymes, which are adapted to high and low temperatures, respectively. These enzymes are widely assumed to also have unique rate temperature dependencies. Thermophilic enzymes, for example, are considered optimal at high temperatures and effectively inactive at low temperatures due to excess rigidity. Surveying published data, we find that thermophilic, mesophilic, and psychrophilic enzymes exhibit indistinguishable rate temperature dependencies. Furthermore, given the nonenzymatic rate temperature dependency, all enzymes, regardless of their operation temperatures, become >10-fold less powerful catalysts per 25 C temperature increase. Among other factors, this loss of rate acceleration may be ascribed to thermally induced vibrations compromising the activesite catalytic configuration, suggesting that many enzymes are in fact insufficiently rigid.
Protein engineering by directed evolution relies on the use of libraries enriched with beneficial variants. Such libraries should explore large mutational diversities while avoiding high loads of deleterious mutations. Here we describe a simple protocol for incorporating synthetic oligonucleotides that encode designed, site-specific mutations by assembly PCR. This protocol enables a researcher to "hedge the bets," namely, to explore a large number of potentially beneficial mutations in a combinatorial manner such that individual library variants carry a limited number of mutations.
VX and its Russian (RVX) and Chinese (CVX) analogues rapidly inactivate acetylcholinesterase and are the most toxic stockpile nerve agents. These organophosphates have a thiol leaving group with a choline-like moiety and are hydrolyzed very slowly by natural enzymes. We used an integrated computational and experimental approach to increase Brevundimonas diminuta phosphotriesterase's (PTE) detoxification rate of V-agents by 5000-fold. Computational models were built of the complex between PTE and V-agents. On the basis of these models, the active site was redesigned to be complementary in shape to VX and RVX and to include favorable electrostatic interactions with their choline-like leaving group. Small libraries based on designed sequences were constructed. The libraries were screened by a direct assay for V-agent detoxification, as our initial studies showed that colorimetric surrogates fail to report the detoxification rates of the actual agents. The experimental results were fed back to improve the computational models. Overall, five rounds of iterating between experiment and model refinement led to variants that hydrolyze the toxic S-P isomers of all three V-agents with k(cat)/K-m values of up to 5 X 10(6) M-1 min(-1) and also efficiently detoxify G-agents. These new catalysts provide the basis for broad spectrum nerve agent detoxification.
Short insertions and deletions (InDels) comprise an important part of the natural mutational repertoire. InDels are, however, highly deleterious, primarily because two-thirds result in frame-shifts. Bypass through slippage over homonucleotide repeats by transcriptional and/or translational infidelity is known to occur sporadically. However, the overall frequency of bypass and its relation to sequence composition remain unclear. Intriguingly, the occurrence of InDels and the bypass of frame-shifts are mechanistically related - occurring through slippage over repeats by DNA or RNA polymerases, or by the ribosome, respectively. Here, we show that the frequency of frame-shifting InDels, and the frequency by which they are bypassed to give full-length, functional proteins, are indeed highly correlated. Using a laboratory genetic drift, we have exhaustively mapped all InDels that occurred within a single gene. We thus compared the naive InDel repertoire that results from DNA polymerase slippage to the frame-shifting InDels tolerated following selection to maintain protein function. We found that InDels repeatedly occurred, and were bypassed, within homonucleotide repeats of 3-8 bases. The longer the repeat, the higher was the frequency of InDels formation, and the more frequent was their bypass. Besides an expected 8A repeat, other types of repeats, including short ones, and G and C repeats, were bypassed. Although obtained in vitro, our results indicate a direct link between the genetic occurrence of InDels and their phenotypic rescue, thus suggesting a potential role for frame-shifting InDels as bridging evolutionary intermediates.
Serum paraoxonases (PONs) are detoxifying lactonases that were first identified in mammals. Three mammalian families are known, PON1, 2, and 3 that reside primarily in the liver. They catalyze essentially the same reaction, lactone hydrolysis, but differ in their substrate specificity. Although some members are highly specific, others have a broad specificity profile. The evolutionary origins and substrate specificities of PONs therefore remain poorly understood. Here, we report a newly identified family of bacterial PONs, and the reconstruction of the ancestor of the three families of mammalian PONs. Both the mammalian ancestor and the characterized bacterial PONX_OCCAL were found to efficiently hydrolyze N-acyl homoserine lactones that mediate quorum sensing in many bacteria, including pathogenic ones. The mammalian PONs may therefore relate to a newly identified family of bacterial, PON-like "quorum-quenching" lactonases. The appearance of PONs in metazoa is likely to relate to innate immunity rather than detoxification. Unlike the bacterial PON, the mammalian ancestor also hydrolyzes, with low efficiency, lactones other than homoserine lactones, thus preceding the detoxifying functions that diverged later in two of the three mammalian families. The bifunctionality of the mammalian ancestor and the trade-off between the quorum-quenching and detoxifying lactonase activities explain the broad and overlapping specificities of some mammalian PONs versus the singular specificity of others.
Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase (PGK), where lysine 219 is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs. Contemporary sequences tested exhibited complete loss of function upon exchanges at 219. However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position 219. These two mutations made position 219 permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at 219 show marked sign epistasis - a relatively small loss of function with respect to one amino acid (lysine) versus a large gain with another (serine, and other amino acids). Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled K219S exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change.
Protein evolvability includes two elements-robustness (or neutrality, mutations having no effect) and innovability (mutations readily inducing new functions). How are these two conflicting demands bridged? Does the ability to bridge them relate to the observation that certain folds, such as TIM barrels, accommodate numerous functions, whereas other folds support only one? Here, we hypothesize that the key to innovability is polarity-an active site composed of flexible, loosely packed loops alongside a well-separated, highly ordered scaffold. We show that highly stabilized variants of TEM-1 beta-lactamase exhibit selective rigidification of the enzyme's scaffold while the active-site loops maintained their conformational plasticity. Polarity therefore results in stabilizing, compensatory mutations not trading off, but instead promoting the acquisition of new activities. Indeed, computational analysis indicates that in folds that accommodate only one function throughout evolution, for example, dihydrofolate reductase, >= 60% of the active-site residues belong to the scaffold. In contrast, folds associated with multiple functions such as the TIM barrel show high scaffold-active-site polarity (similar to 20% of the active site comprises scaffold residues) and >2-fold higher rates of sequence divergence at active-site positions. Our work suggests structural measures of fold polarity that appear to be correlated with innovability, thereby providing new insights regarding protein evolution, design, and engineering. (C) 2013 Elsevier Ltd. All rights reserved.
Recent advances in enzyme engineering enable dramatic improvements in catalytic efficiency and/or selectivity, as well as de novo engineering of enzymes to catalyze reactions where natural enzymes are not available. Can these capabilities be utilized to transform biosynthesis pathways? Metabolic engineering is traditionally based on combining existing enzymes to give new, or modified, pathways, within a new context and/or organism. How efficient, however, are the individual enzyme components? Is there room to improve pathway performance by enzyme engineering? We discuss the differences between enzymes in central versus specialized, or secondary metabolism and highlight unique features of specialized metabolism enzymes participating in the synthesis of natural products. We argue that, for the purpose of metabolic engineering, the catalytic efficiency and selectivity of many enzymes can be improved with the aim of achieving higher rates, yields and product purities. We also note the relative abundance of spontaneous reactions in specialized metabolism, and the potential advantage of engineering enzymes that will catalyze these steps. Specialized metabolism therefore offers new opportunities to integrate enzyme and pathway engineering, thereby achieving higher metabolic efficiencies, enhanced production rates and improved product purities.
Backbone modifications via insertions and deletions (InDels) may exert dramatic effects, for better (mediating new functions) and for worse (causing loss of structure and/or function). However, contrary to point mutations (substitutions), our knowledge of the evolution and structural-functional effects of InDels is limited and so is our capability to engineer them. We sought to assess how deleterious InDels are relative to point mutations and understand the mechanisms that mediate their acceptance. Analysis of the evolution of InDels in orthologous protein phylogenies indicated that their rate of purging is 9- to 100-fold higher than for point mutations. In yeast, for example, the substitutions-to-InDels ratio is approximately 14-fold higher in protein coding than in noncoding regions. The incorporation of InDels relative to substitutions is not only slow but also nonlinear. On average, epsilon 50 substitutions accumulate before the appearance of the first InDel. We also found enriched substitutions in sequential and spatial proximity to InDels, suggesting that certain substitutions are correlated with InDels. As indicated by the lag in InDels accumulation, some of these correlated substitutions may have occurred first, as apparently neutral mutations, and later enabled the accumulation of InDels that would be otherwise purged. Thus, compensatory substitutions may follow InDels in an "adaptive walk" as traditionally assumed, but might also accumulate first, by "neutral roaming." The dynamics of InDels accumulation also depends on their genomic frequencies-InDels in flies are 4-fold more frequent than in yeast and tend to be compensated rather than enabled.
Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca2+. We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gin indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-angstrom upward displacement towards the enzyme's surface of the catalytic Ca2+ in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca2+ mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions. (C) 2013 Elsevier Ltd. All rights reserved.
This review outlines the strategies we apply for directed enzyme evolution using targeted libraries, namely, libraries that diversify specific residues with predefined mutational compositions. The theoretical grounds underlining the design of such libraries are described, including the mutational load, the ratio of beneficial versus deleterious mutations, and screening capacity. We point out the advantage of using mutational spiking strategies for "hedging the bets," exploring a large number of potentially beneficial mutations, and tuning the library's mutational load. Also highlighted are the merits of low-throughput screens that measure multiple parameters at high accuracy, and of using the desired substrate and reaction conditions rather than surrogates. We subsequently describe library construction strategies (rational and analytical) based on structure and sequence analyses, including ancestral libraries, which are particularly suitable for low-throughput screens. We also discuss the critical role of including compensatory, stabilizing mutations during library construction. Finally, the design efficiency and the optimal mutational loads of libraries are assessed by comparing targeted mutational libraries versus libraries of random mutations.
DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M. HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M. HaeIII [GG((A)/(T))CC and GGCG CC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M. HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.
Optimization processes, such as evolution, are constrained by diminishing returns-the closer the optimum, the smaller the benefit per mutation, and by tradeoffs-improvement of one property at the cost of others. However, the magnitude and molecular basis of these parameters, and their effect on evolutionary transitions, remain unknown. Here we pursue a complete functional transition of an enzyme with a >10(9)-fold change in the enzyme's selectivity using laboratory evolution. We observed strong diminishing returns, with the initial mutations conferring >25-fold higher improvements than later ones, and asymmetric tradeoffs whereby the gain/loss ratio of the new/old activity decreased 400-fold from the beginning of the trajectory to its end. We describe the molecular basis for these phenomena and suggest they have an important role in shaping natural proteins. These findings also suggest that the catalytic efficiency and specificity of many natural enzymes may be far from their optimum.
Gene expression depends on the frequency of transcription events (burst frequency) and on the number of mRNA molecules made per event (burst size). Both processes are encoded in promoter sequence, yet their dependence on mutations is poorly understood. Theory suggests that burst size and frequency can be distinguished by monitoring the stochastic variation (noise) in gene expression: Increasing burst size will increase mean expression without changing noise, while increasing burst frequency will increase mean expression and decrease noise. To reveal principles by which promoter sequence regulates burst size and frequency, we randomly mutated 22 yeast promoters chosen to span a range of expression and noise levels, generating libraries of hundreds of sequence variants. In each library, mean expression (m) and noise (coefficient of variation, eta) varied together, defining a scaling curve: eta(2) = b/m + eta(2)(ext). This relation is expected if sequence mutations modulate burst frequency primarily. The estimated burst size (b) differed between promoters, being higher in promoter containing a TATA box and lacking a nucleosome-free region. The rare variants that significantly decreased b were explained by mutations in TATA, or by an insertion of an out-of-frame translation start site. The decrease in burst size due to mutations in TATA was promoter-dependent, but independent of other mutations. These TATA box mutations also modulated the responsiveness of gene expression to changing conditions. Our results suggest that burst size is a promoter-specific property that is relatively robust to sequence mutations but is strongly dependent on the interaction between the TATA box and promoter nucleosomes.
Arsenate and phosphate are abundant on Earth and have striking similarities: nearly identical pK(a) values(1,2), similarly charged oxygen atoms, and thermochemical radii that differ by only 4% (ref. 3). Phosphate is indispensable and arsenate is toxic, but this extensive similarity raises the question whether arsenate may substitute for phosphate in certain niches(4,5). However, whether it is used or excluded, discriminating phosphate from arsenate is a paramount challenge. Enzymes that utilize phosphate, for example, have the same binding mode and kinetic parameters as arsenate, and the latter's presence therefore decouples metabolism(6,7). Can proteins discriminate between these two anions, and how would they do so? In particular, cellular phosphate uptake systems face a challenge in arsenate-rich environments. Here we describe a molecular mechanism for this process. We examined the periplasmic phosphate-binding proteins (PBPs) of the ABC-type transport system that mediates phosphate uptake into bacterial cells, including two PBPs from the arsenate-rich Mono Lake Halomonas strain GFAJ-1. All PBPs tested are capable of discriminating phosphate over arsenate at least 500-fold. The exception is one of the PBPs of GFAJ-1 that shows roughly 4,500-fold discrimination and its gene is highly expressed under phosphate-limiting conditions. Sub-angstrom-resolution structures of Pseudomonas fluorescens PBP with both arsenate and phosphate show a unique mode of binding that mediates discrimination. An extensive network of dipole-anion interactions(8,9), and of repulsive interactions, results in the 4% larger arsenate distorting a unique low-barrier hydrogen bond. These features enable the phosphate transport system to bind phosphate selectively over arsenate (at least 10(3) excess) even in highly arsenate-rich environments.
In nature, the evolution of new protein functions is driven not only by side-chain substitutions (point mutations), but also by backbone modifications (insertions and deletions). The current laboratory diversification methods, however, are largely limited to point mutations. Of particular interest are short insertions-by-duplication that are frequent in nature but cannot be introduced in vitro in a library format (i.e. in random locations and lengths). Here, we describe a new procedure that allows the generation of tandem repeats of random fragments of the target gene via rolling-circle amplification, and the concurrent incorporation of these repeats into the target gene. This procedure, dubbed tandem repeat insertion, or TRINS, results in a library of genes carrying insertions-by-duplication of variable lengths (3150 bp) at random positions. This diversification pattern allows sampling of sequence space regions that are not readily accessible by other protocols. We demonstrate this method by constructing three different gene libraries, and by selecting insertion variants of TEM-1 -lactamase.
The field of directed evolution has progressed to the point where it is feasible to engineer enzymes for unnatural substrates and reactions with catalytic efficiencies and regio-specificity or stereo-specificity that rival those of natural enzymes. Here, we describe the conceptual and methodological advances that have enabled this progress. We address methodologies based on small libraries enriched with improved variants and carrying compensatory stabilizing mutations. Such libraries can be combined with low-throughput screens that provide high accuracy and directly target the desired substrate and reaction conditions, and thereby provide highly improved variants.
Only decades after the introduction of organophosphate pesticides, bacterial phosphotriesterases (PTEs) have evolved to catalyze their degradation with remarkable efficiency. Their closest known relatives, lactonases, with promiscuous phosphotriasterase activity, dubbed PTE-like lactonases (PLLs), share only 30% sequence identity and also differ in the configuration of their active-site loops. PTE was therefore presumed to have evolved from a yet unknown PLL whose primary activity was the hydrolysis of quorum sensing homoserine lactones (HSLs) (Afriat et al. (2006) Biochemistry 45, 13677-13686). However, how PTEs diverged from this presumed PLL remains a mystery. In this study we investigated loop remodeling as a means of reconstructing a homoserine lactonase ancestor that relates to PTE by few mutational steps. Although, in nature, loop remodeling is a common mechanism of divergence of enzymatic functions, reproducing this process in the laboratory is a challenge. Structural and phylogenetic analyses enabled us to remodel one of PTE's active-site loops into a PLL-like configuration. A deletion in loop 7, combined with an adjacent, highly epistatic, point mutation led to the emergence of an HSLase activity that is undetectable in PTE (k(cat)/K-M values of up to 2 X 10(4)). The appearance of the HSLase activity was accompanied by only a minor decrease in PTE's paraoxonase activity. This specificity change demonstrates the potential role of bifunctional intermediates in the divergence of new enzymatic functions and highlights the critical contribution of loop remodeling to the rapid divergence of new enzyme functions.
Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K-m values of 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K-m values up to 0.6 x 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of
The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1. (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential. (C) 2012 Elsevier Ltd. All rights reserved.
A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S-P isomers of the most threatening G-type nerve agents. The evolved variants show 2,500 for the S-P isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/k(m)) of -10(4) M(-1)s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R-p isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
We discuss the basic features of divergent versus convergent evolution and of the common scenario of parallel evolution. The example of quorum-quenching lactonases is subsequently described. Three different quorum-quenching lactonase families are known, and they belong to three different superfamilies. Their key active-site architectures have converged and are strikingly similar. Curiously, a promiscuous organophosphate hydrolase activity is observed in all three families. We describe the structural and mechanistic features that underline this converged promiscuity and how this promiscuity drove the parallel divergence of organophosphate hydrolases within these lactonase families by either natural or laboratory evolution.
An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k(cat)/K(m) value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
Large libraries of randomly mutated genes are applied in directed evolution experiments in order to obtain sufficient variability. These libraries, however, contain mostly inactive variants, and the very low frequency of improved variants can only be isolated by high-throughput screening. Small but efficient libraries comprise an attractive alternative. Here, we describe the application of ancestral libraries-libraries based on mutations predicted by phylogenetic analysis and ancestral inference. We designed and constructed such libraries using serum paraoxonases and cytosolic sulfotransferases (SULTs) as model enzymes. Both of these enzyme families exhibit a range of activities in drug metabolism and detoxification of xenobiotics. The ancestral serum paraoxonase and SULT libraries were screened by low-throughput means, including HPLC, using substrates and reactions with which all family members exhibit low activity. The libraries showed a remarkably high frequency of highly polymorphic and functionally diverse variants. Screening of as few as 300 variants enabled the isolation of mutants with up to 50-fold higher activity than the starting point enzyme. Structural and kinetic characterizations of an evolved SULT variant show how few ancestral mutations reshaped the active site and modulated the enzyme's specificity. Ancestral libraries therefore comprise a means of focusing diversity to positions and mutations that readily trigger changes in substrate and/or reaction specificity, thereby facilitating the isolation of new enzyme variants for a variety of different substrates and reactions by medium-throughput or even low-throughput screens. (C) 2011 Elsevier Ltd. All rights reserved.
Why do certain proteins evolve much slower than others? We compared not only rates per protein, but also rates per position within individual proteins. For similar to 90% of proteins, the distribution of positional rates exhibits three peaks: a peak of slow evolving residues, with average log(2)[normalized rate], log(2)mu, of ca. -2, corresponding primarily to core residues; a peak of fast evolving residues (log(2)mu similar to 0.5) largely corresponding to surface residues; and a very fast peak (log(2)mu similar to 2) associated with disordered segments. However, a unique fraction of proteins that evolve very slowly exhibit not only a negligible fast peak, but also a peak with a log(2)mu similar to-4, rather than the standard core peak of -2. Thus, a "freeze" of a protein's surface seems to stop core evolution as well. We also observed a much higher fraction of substitutions in potentially interacting residues than expected by chance, including substitutions in pairs of contacting surface-core residues. Overall, the data suggest that accumulation of surface substitutions enables the acceptance of substitutions in core positions. The underlying reason for slow evolution might therefore be a highly constrained surface due to protein-protein interactions or the need to prevent misfolding or aggregation. If the surface is inaccessible to substitutions, so becomes the core, thus resulting in very slow overall rates.
The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of k(cat) and K-M values of several thousand enzymes collected from the literature. We found that the "average enzyme" exhibits a k(cat) of similar to 10 s(-1) and a k(cat)/K-M of similar to 10(5) s(-1) M-1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, similar to 30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit K-M optimization. In accordance, substitution with phosphate, Colt, or other large modifiers considerably lowers the K-M values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochemical constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or laboratory evolution.
Two different scenarios for the recruitment of evolutionary starting points and their subsequent divergence to give new enzymes have been described. The coincidental, promiscuous starting activity may regard the same reaction chemistry on a new substrate (substrate ambiguity). Alternatively, substrate binding guides the recruitment of an enzyme whose reaction chemistry differs from that of the newly evolving one (catalytic promiscuity). While substrate ambiguity seems to underlie the divergence of most enzyme families, the relative levels of occurrence of these scenarios remain unknown. Screening the Escherichia coli proteome with a comparative series of xenobiotic substrates, we found that substrate ambiguity was, as anticipated, more frequent than reaction promiscuity. However, for at least one unnatural reaction (phosphonoesterase), a promiscuous enzyme was identified only when the substrate was decorated with the naturally abundant phosphate group. These findings support the prevailing hypothesis of chemistry-driven divergence but also suggest that recognition of familiar substrate motifs plays a role. In the absence of enzymes catalyzing the same chemistry, having a familiar, naturally occurring substrate motif (chemophore) such as phosphate may increase the likelihood of catalytic promiscuity. Chemophore anchoring may also find practical applications in identifying catalysts for unnatural reactions.
Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5 x 10(4) s(-1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis. (C) 2011 Elsevier Ltd. All rights reserved.
Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 beta-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.
A newly identified bacterial strain that can grow in the presence of arsenate and possibly in the absence of phosphate, has raised much interest, but also fueled an active debate. Can arsenate substitute for phosphate in some or possibly in most of the absolutely essential phosphate-based biomolecules, including DNA? If so, then the possibility of alternative, arsenic-based life forms must be considered. The physicochemical similarity of these two oxyanions speaks in favor of this idea. However, arsenate-esters and arsenate-diesters in particular are extremely unstable in aqueous media. Here, we explore the potential of arsenate to be used as substrate by phosphate-utilizing enzymes. We review the existing literature on arsenate enzymology, that intriguingly, dates back to the 1930s. We address the issue of how and to what degree proteins can distinguish between arsenate and phosphate and what is known in general about oxyanion specificity. We also discuss how phosphate arsenate promiscuity may affect evolutionary transitions between phosphate- and arsenate-based biochemistry. Finally, we highlight potential applications of arsenate as a structural and mechanistic probe of enzymes whose catalyzed reactions involve the making or breaking of phosphoester bonds.
Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by similar to 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) similar to 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
Internal symmetry in proteins is likely to be the footprint of evolution by gene duplication and fusion. Like other symmetrical proteins, beta-propellers, which are made of 4-10 beta-sheet units (blades) circularly arranged around a central tunnel, have probably evolved by duplication and fusion of a rudimentary repetitive unit. However, reproducing the evolution of functional beta-propellers by duplication and fusion of repeated units remains a challenge, in particular, because the repeated units must jointly pack to form one hydrophobic core while maintaining intact active sites. As model for generating repeat propellers, we chose tachylectin-2-a highly symmetrical five-bladed beta-propeller lectin with five sugar-binding sites. We report the engineering of folded and functional lectins by duplication and fusion of repetitive sequence modules taken from tachylectin-2. The repeated modules comprise three strands of one blade plus one strand of the next blade, thus enabling the closure of the propeller's ring via strand-strand Velcro-like interactions. Duplication and fusion of five modules with the same sequence gave rise to a highly aggregated protein, yet its soluble fraction exhibited lectin function. Subsequently, a library of diversified sequence modules fused in tandem was selected by phage display for glycoprotein binding. A range of new lectins were isolated with binding and biophysical properties that resemble those of wild-type tachylectin-2. These results demonstrate the ability to construct folded and functional globular repeat proteins, and support the role of duplication and fusion of elementary modules in the evolutionary routes that led to the beta-propellers fold.
Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600-and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >> 1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
The divergence of new genes and proteins occurs through mutations that modulate protein function. However, mutations are pleiotropic and can have different effects on organismal fitness depending on the environment, as well as opposite effects on protein function and dosage. We review the pleiotropic effects of mutations. We discuss how they affect the evolution of gene and protein function, and how these complex mutational effects dictate the likelihood and mechanism of gene duplication and divergence. We propose several factors that can affect the divergence of new protein functions, including mutational trade-offs and hidden, or apparently neutral, variation.
The primary sequence of proteins usually dictates a single tertiary and quaternary structure. However, certain proteins undergo reversible backbone rearrangements. Such metamorphic proteins provide a means of facilitating the evolution of new folds and architectures. However, because natural folds emerged at the early stages of evolution, the potential role of metamorphic intermediates in mediating evolutionary transitions of structure remains largely unexplored. We evolved a set of new proteins based on similar to 100 amino acid fragments derived from tachylectin-2-a monomeric, 236 amino acids, five-bladed beta-propeller. Their structures reveal a unique pentameric assembly and novel beta-propeller structures. Although identical in sequence, the oligomeric subunits adopt two, or even three, different structures that together enable the pentameric assembly of two propellers connected via a small linker. Most of the subunits adopt a wild-type-like structure within individual five-bladed propellers. However, the bridging subunits exhibit domain swaps and asymmetric strand exchanges that allow them to complete the two propellers and connect them. Thus, the modular and metamorphic nature of these subunits enabled dramatic changes in tertiary and quaternary structure, while maintaining the lectin function. These oligomers therefore comprise putative intermediates via which beta-propellers can evolve from smaller elements. Our data also suggest that the ability of one sequence to equilibrate between different structures can be evolutionary optimized, thus facilitating the emergence of new structures.
Understanding enzyme catalysis through the analysis of natural enzymes is a daunting challenge-their active sites are complex and combine numerous interactions and catalytic forces that are finely coordinated. Study of more rudimentary (wo)man-made enzymes provides a unique opportunity for better understanding of enzymatic catalysis. KE07, a computationally designed Kemp eliminase that employs a glutamate side chain as the catalytic base for the critical proton abstraction step and an apolar binding site to guide substrate binding, was optimized by seven rounds of random mutagenesis and selection, resulting in a >200-fold increase in catalytic efficiency. Here, we describe the directed evolution process in detail and the biophysical and crystallographic studies of the designed KE07 and its evolved variants. The optimization of KE07's activity to give a k(cat)/K(M) value of similar to 2600 s(-1) M(-1) and an similar to 10(6)-fold rate acceleration (k(cat)/k(uncat)) involved the incorporation of up to eight mutations. These mutations led to a marked decrease in the overall thermodynamic stability of the evolved KE07s and in the configurational stability of their active sites. We identified two primary contributions of the mutations to KE07's improved activity: (i) the introduction of new salt bridges to correct a mistake in the original design that placed a lysine for leaving-group protonation without consideration of its "quenching" interactions with the catalytic glutamate, and (ii) the tuning of the environment, the pK(a) of the catalytic base, and its interactions with the substrate through the evolution of a network of hydrogen bonds consisting of several charged residues surrounding the active site. (C) 2010 Elsevier Ltd. All rights reserved.
Serum paraoxonase (PON1) is all anti-atherogenic interfacially activated lipo-lactonase that Was shown to selectively bind high-density lipoprotein (HDL) carrying apolipoprotein A-1 (apoA-1). ApoA-1 binding occurs with nanomolar affinity and Induces a dramatic increase in enzyme stability and lactonase activity. This study examined the association of PON1 With reconstituted HDL (rHDL) carrying apolipoprotein E, and its consequences on the stability and enzymatic activity of PON1, and on its anti-atherogenic potential. The results indicate that reconstituted HDL particles prepared With two most common isoforms of apoE (apoE3 and apoE4) associate with rePON1 in a manner and affinity similar to those of apoA-1. Binding to apoE-HDL stimulates the lactonase activity and stabilizes the enzyme, although the latter Occurs to a > 10-rold lesser extent compared to apoA-I-HDL particles. The anti-atherogenic potential of PON1, measured by inhibition of LDL oxidation and stimulation of macrophage cholesterol efflux, was also stimulated by apoE-HDL, at levels of 40-96% compared to apoA-1-HDL. Overall, reconstituted apoE-HDL exhibits properties similar to those of apoA-1-HDL, but with a lower capacity to stabilize PON1 and to induce its anti-atherogenic functions. ApoE, apoA-1, and to a lesser degree apoA-IV show distinct structural and functional similarities but little sequence homology. That these apolipoproteins. but not apoA-11, bind PON1 with high affinity and stimulate its activity Suggests that PON1-HDL recognition is based primarily oil surface properties of the apolipoproteins and that specific protein-protein interactions may play only a secondary role.
Many, if not most, enzymes can promiscuously catalyze reactions, or act on substrates, other than those for which they evolved. Here, we discuss the structural, mechanistic, and evolutionary implications of this manifestation of infidelity of molecular recognition. We define promiscuity and related phenomena and also address their generality and physiological implications. We discuss the mechanistic enzymology of promiscuity-how enzymes, which generally exert exquisite specificity, catalyze other, and sometimes barely related, reactions. Finally, we address the hypothesis that promiscuous enzymatic activities serve as evolutionary starting points and highlight the unique evolutionary features of promiscuous enzyme functions.
To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open'' and "closed'' CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.
How do intricate multi-residue features such as protein-protein interfaces evolve? To address this question, we evolved a new colicin-immunity binding interaction. We started with Im9, which inhibits its cognate DNase ColE9 at 10(-14) M affinity, and evolved it toward ColE7, which it inhibits promiscuously (K(d) > 10(-8) M). Iterative rounds of random mutagenesis and selection toward higher affinity for ColE7, and selectivity ( against ColE9 inhibition), led to an similar to 10(5)-fold increase in affinity and a 10(8)-fold increase in selectivity. Analysis of intermediates along the evolved variants revealed that changes in the binding configuration of the Im protein uncovered a latent set of interactions, thus providing the key to the rapid divergence of new Im7 variants. Overall, protein-protein interfaces seem to share the evolvability features of enzymes, that is, the exploitation of promiscuous interactions and alternative binding configurations via 'generalist' intermediates, and the key role of compensatory stabilizing mutations in facilitating the divergence of new functions.
The past several years have seen novel insights at the interface of protein biophysics and evolution. The accepted paradigm that proteins can tolerate nearly any amino acid substitution has been replaced by the view that the deleterious effects of mutations, and especially their tendency to undermine the thermodynamic and kinetic stability of protein, is a major constraint on protein evolvability-the ability of proteins to acquire changes in sequence and function. We summarize recent findings regarding how mutations affect protein stability, and how stability affects protein evolution. We describe ways of predicting and analyzing stability effects of mutations, and mechanisms that buffer or compensate for these destabilizing effects and thereby promote protein evolvabilty, in nature and in the laboratory.
Serum paraoxonases (PONs) are calcium-dependent lactonases with anti-atherogenic and detoxification functions. Here we describe the directed evolution and characterization of recombinant variants of serum paraoxonase PON3 that express in an active and soluble manner in Escherichia coli. These variants were obtained by combining family shuffling and phylogeny-based mutagenesis: the limited diversity of accessible, cloned PON3 genes was complemented by spiking the shuffling reaction with ancestor/consensus mutations, mutations to residues that comprise the consensus or appear in the predicted ancestors of the PON family, We screened the resulting libraries for PON3's lactonase activity while ensuring that the selected variants retained the Substrate specificity of wild-type mammalian PON3s. The availability of highly stable, recombinant PON3 that is free of all other serum components enabled us to explore unknown biochemical features of PON3, including its binding to HDL particles, the effect of HDL on PON3's stability and enzymatic activity, and ex vivo tests of its anti-atherogenic properties. Overall, it appears that PON3 possesses properties very similar to those of PON I: the enzyme's lactonase activity is selectively stimulated by binding to apoAI-HDL, with a concomitant increase in its stability. PON3 also exhibits potentially anti-atherogenic functions, although at levels lower than those of PON1.
Most protein mutations, and mutations that alter protein functions in particular, undermine stability and are therefore deleterious. Chaperones, or heat-shock proteins, are often implicated in buffering mutations, and could thus facilitate the acquisition of neutral genetic diversity and the rate of adaptation. We examined the ability of the Escherichia coli GroEL/GroES chaperonins to buffer destabilizing and adaptive mutations. Here we show that mutational drifts performed in vitro with four different enzymes indicated that GroEL/GroES overexpression doubled the number of accumulating mutations, and promoted the folding of enzyme variants carrying mutations in the protein core and/or mutations with higher destabilizing effects (destabilization energies of >3.5 kcal mol(-1), on average, versus, similar to 1 kcal mol(-1) in the absence of GroEL/GroES). The divergence of modified enzymatic specificity occurred much faster under GroEL/GroES overexpression, in terms of the number of adapted variants (>= 2-fold) and their improved specificity and activity (>= 10-fold). These results indicate that protein stability is a major constraint in protein evolution, and buffering mechanisms such as chaperonins are key in alleviating this constraint.
The traditional view that proteins possess absolute functional specificity and a single, fixed structure conflicts with their marked ability to adapt and evolve new functions and structures. We consider an alternative, "avant-garde view" in which proteins are conformationally dynamic and exhibit functional promiscuity. We surmise that these properties are the foundation stones of protein evolvability; they facilitate the divergence of new functions within existing folds and the evolution of entirely new folds. Packing modes of proteins also affect their evolvability, and poorly packed, disordered, and conformationally diverse proteins may exhibit high evolvability. This dynamic view of protein structure, function, and evolvability is extrapolated to describe hypothetical scenarios for the evolution of the early proteins and future research directions in the area of protein dynamism and evolution.
Natural selection shapes the sequence, structure and biophysical properties of proteins to fit their environment. We hypothesize that highly thermostable proteins and viral proteins represent two opposing adaptation strategies. Thermostable proteins are highly compact and possess well-packed hydrophobic cores and intensely charged surfaces. By contrast, viral proteins, and RNA viral proteins in particular, display a high occurrence of disordered segments and loosely packed cores. These features might endow viral proteins with increased structural flexibility and effective ways to interact with the components of the host. They could also be related to high adaptability levels and mutation rates observed in viruses, thus, representing a unique strategy for buffering the deleterious effects of mutations, such that those that have little (interactions), have little to lose.
The divergence of new gene functions is described by various scenarios that involve gene duplication, albeit, at fundamentally different stages. We performed experimental measurements and developed a subsequent model, aimed at predicting the rate of divergence under different scenarios. We used gene libraries of TEM-1 beta-lactamase that were drifted under purifying selection toward the original penicillinase activity or under no selection at all. The frequency of genes conferring a new function (degradation of a cephalosporin antibiotic) was measured at various stages of the drift, and a model that accounts for the differences in the observed adaptation dynamics of the drifting TEM-1 populations was derived. The results indicate that rapid nonfunctionalization in the population relieved from selection (Ohno's model) afforded only a narrow window of adaptation to cefotaxime (neofunctionalization). The trade-off between TEM-1's original function and the new evolving function also disfavored the "gene sharing" model. The rate of adaptation was maximal when selection for the original function was partially relieved to enable the accumulation of potentially adaptive mutations, while still purging a large fraction of otherwise deleterious mutations. Altogether, scenarios of subfunctionalization seem more feasible, whereby sustaining the original function by two copies facilitates the accumulation potentially adaptive mutations while purging nonfunctionalization mutations.
Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.
What changes occur when a natural protein that had been under low mutation rates is subjected to a neutral drift at high mutational loads, thus generating genetically diverse (polymorphic) gene ensembles that all maintain the protein's original function and structure? To address this question we subjected large populations of TEM-1 beta-lactamase to a prolonged neutral drift, applying high mutation rates and purifying selection to maintain TEM-1's existing penicillinase activity. Purging of deleterious mutations and enrichment of beneficial ones maintained the sequence of these ensembles closer to TEM-1's family consensus and inferred ancestor. In particular, back-to-consensus/ancestor mutations that increase TEM-1's kinetic and thermodynamic stability were enriched. These acted as global suppressors and enabled the tolerance of a broad range of deleterious mutations, thus further increasing the genetic diversity of the drifting populations. The probability of a new function emerging (cefotaxime degradation) was also substantially increased in these ensembles owing to the presence of many gene variants carrying the global suppressors. Our findings indicate the unique features of large, polymorphic neutral ensembles generated under high mutational loads and prompt the speculation that the progenitors of today's proteins may have evolved under high mutational loads. The results also suggest that predictable back-to-consensus/ancestor changes can be used in the laboratory to generate highly diverse and evolvable gene libraries. (C) 2008 Elsevier Ltd. All rights reserved.
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination - a model reaction for proton transfer from carbon - with measured rate enhancements of up to 10 5 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high- resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K-m (k(cat)/K-m of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FAGS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.
Numerous studies have noted that the evolution of new enzymatic specificities is accompanied by loss of the protein's thermodynamic stability (Delta Delta G), thus suggesting a tradeoff between the acquisition of new enzymatic functions and stability. However, since most mutations are destabilizing (Delta Delta G>0), one should ask how destabilizing mutations that confer new or altered enzymatic functions relative to all other mutations are. We applied Delta Delta G computations by FoldX to analyze the effects of 548 mutations that arose from the directed evolution of 22 different enzymes. The stability effects, location, and type of function-altering mutations were compared to Delta Delta G changes arising from all possible point mutations in the same enzymes. We found that mutations that modulate enzymatic functions are mostly destabilizing (average Delta Delta G = +0.9 kcal/mol), and are almost as destabilizing as the "average" mutation in these enzymes (+1.3 kcal/mol). Although their stability effects are not as dramatic as in key catalytic residues, mutations that modify the substrate binding pockets, and thus mediate new enzymatic specificities, place a larger stability burden than surface mutations that underline neutral, non- adaptive evolutionary changes. How are the destabilizing effects of functional mutations balanced to enable adaptation? Our analysis also indicated that many mutations that appear in directed evolution variants with no obvious role in the new function exert stabilizing effects that may compensate for the destabilizing effects of the crucial function-altering mutations. Thus, the evolution of new enzymatic activities, both in nature and in the laboratory, is dependent on the compensatory, stabilizing effect of apparently "silent" mutations in regions of the protein that are irrelevant to its function.
When generating novel tailor-made proteins, protein engineers routinely apply the principles of 'Darwinian' evolution. However, laboratory evolution of proteins also has the potential to test evolutionary theories and reproduce evolutionary scenarios, thus reconstructing putative protein intermediates and providing a glimpse of 'protein fossils'. This commentary describes research at the interface of applied and fundamental molecular evolution, and provides a personal view of how synergy between fundamental and applied experiments indicates novel and more efficient ways of generating new proteins in the laboratory.
This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants'-i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.
Serum paraoxonase (PON1) is a lipolactonase that associates with HDL-apolipoprotein A-I (HDL-apoA-I) and thereby plays a role in the prevention of atherosclerosis. Current sera tests make use of promiscuous substrates and provide no indications regarding HDL-PON1 complex formation. We developed new enzymatic tests that detect total PON1 levels, irrespective of HDL status and R/Q polymorphism, as well as the degree of catalytic stimulation and increased stability that follow PON1's tight binding to HDLapoA-I. The tests are based on measuring total PON1 levels with a fluorogenic phosphotriester, measuring the lipolactonase activity with a chromogenic lactone, and assaying the enzyme's chelator-mediated inactivation rate. The latter two are affected by tight HDL binding and thereby derive the levels of the serum PON1-HDL complex. We demonstrate these new tests with a group of healthy individuals (n 5 54) and show that the levels of PON1-HDL vary by a factor of 12. Whereas the traditionally applied paraoxonase and arylesterase tests weakly reflect PON1-HDL levels (R = 0.64), the lipolactonase test provides better correlation (R = 0.80). These new tests indicate the levels and activity of PON1 in a physiologically relevant context as well as the levels and quality of the HDL particles with which the enzyme is associated.
How the thermodynamic stability effects of protein mutations (Delta Delta G) are distributed is a fundamental property related to the architecture, tolerance to mutations (mutational robustness), and evolutionary history of proteins. The stability effects of mutations also dictate the rate and dynamics of protein evolution, with deleterious mutations being the main inhibitory factor. Using the FoldX algorithm that attempts to computationally predict Delta Delta G effects of mutations, we deduced the overall distributions of stability effects for all possible mutations in 21 different globular, single domain proteins. We found that these distributions are strikingly similar despite a range of sizes and folds, and largely follow a bi-Gaussian function: The surface residues exhibit a narrow distribution with a mildly destabilizing mean Delta Delta G (similar to 0.6 kcal/mol), whereas the core residues exhibit a wider distribution with a stronger destabilizing mean (similar to 1.4 kcal/mol). Since smaller proteins have a higher fraction of surface residues, the relative weight of these single distributions correlates with size. We also found that proteins evolved in the laboratory follow an essentially identical distribution, whereas de novo designed folds show markedly less destabilizing distributions (i.e. they seem more robust to the effects of mutations). This bi-Gaussian model provides an analytical description of the predicted distributions of mutational stability effects. It comprises a novel tool for analyzing proteins and protein models, for simulating the effect of mutations under evolutionary processes, and a quantitative description of mutational robustness. (C) 2007 Elsevier Ltd. All rights reserved.
Biological systems exhibit mutational robustness, or neutrality, whereby the impact of mutations is minimized. Does neutrality hamper their ability to adapt in the face of changing environments? We monitored changes in genotype and phenotype that occur within a neutral mutational network of an enzyme, experimentally and computationally, see accompanying article.. Using the enzyme PON1 as a model, we performed random mutagenesis and purifying selection to purge deleterious mutations. We characterized similar to 300 variants that are apparently neutral, or close to neutral, with respect to PON1's levels of expression and native lactonase activity. Their activities with promiscuous substrates and ligands indicated significant changes in adaptive potentials. Almost half of the variants exhibited changes in promiscuous activities, specificities, or inhibition, and several of these were found to be one or two mutations, closer to potentially new phenotypes: aryl esterase, thiolactonase, phosphotriesterase, or drug resistance. This empirical measure of phenotypic changes under neutrality supports the notion that sequence changes that are neutral, i.e., non-adaptive, in a current context can facilitate adaptation under changing circumstances, by both expanding the activity range of existing enzymes and thus providing an immediate advantage, and by reducing the number of mutations required for divergence of new functions.
The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wildtype gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of. diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.
The modular nature of protein folds suggests that present day proteins evolved via duplication and recombination of smaller functional elements. However, the reconstruction of these putative evolutionary pathways after many millions of years of evolutionary drift has thus far proven difficult, with all attempts to date failing to produce a functional protein. Tachylecin-2 is a monomeric 236 amino acid, five-bladed beta-propeller with five sugar-binding sites. This protein was isolated from a horseshoe crab that emerged ca 500 million years ago. The modular, yet ancient, nature of Tachylectin-2 makes it an excellent model for exploring the evolution of proteins from smaller subunits. To this end, we generated genetically diverse libraries by incremental truncation of the Tachylectin-2 gene and screened them for functional lectins. A number of similar to 100 amino acid residue segments were isolated with the ability to assemble into active homo-pentamers. The topology of most of these segments follows a "hidden" module that differs from the modules observed in wild-type Tachylectin-2, yet their biophysical properties and sugar binding activities resemble the wild-type's. Since the pentamer's molecular mass is twofold higher than the wild-type (similar to 500 amino acid residues), the structure of these oligomeric forms is likely to also differ. Our laboratory evolution experiments highlight the versatility and modularity of the beta-propeller fold, while substantiating the hypothesis that proteins with high internal symmetry such as beta-propellers, evolved from short, functional gene segments that, at later stages, duplicated, fused, and rearranged, to yield the folds we recognise today.
The distribution of fitness effects of protein mutations is still unknown(1,2). Of particular interest is whether accumulating deleterious mutations interact, and how the resulting epistatic effects shape the protein's fitness landscape. Here we apply a model system in which bacterial fitness correlates with the enzymatic activity of TEM-1 beta-lactamase ( antibiotic degradation). Subjecting TEM-1 to random mutational drift and purifying selection ( to purge deleterious mutations) produced changes in its fitness landscape indicative of negative epistasis; that is, the combined deleterious effects of mutations were, on average, larger than expected from the multiplication of their individual effects. As observed in computational systems(3-5), negative epistasis was tightly associated with higher tolerance to mutations ( robustness). Thus, under a low selection pressure, a large fraction of mutations was initially tolerated ( high robustness), but as mutations accumulated, their fitness toll increased, resulting in the observed negative epistasis. These findings, supported by FoldX stability computations of the mutational effects(6), prompt a new model in which the mutational robustness ( or neutrality) observed in proteins, and other biological systems, is due primarily to a stability margin, or threshold, that buffers the deleterious physico-chemical effects of mutations on fitness. Threshold robustness is inherently epistatic - once the stability threshold is exhausted, the deleterious effects of mutations become fully pronounced, thereby making proteins far less robust than generally assumed.
In essence, evolutionary processes occur gradually, while maintaining fitness throughout. Along this line, it has been proposed that the ability of a progenitor to promiscuously catalyze a low level of the evolving activity could facilitate the divergence of a new function by providing an immediate selective advantage. To directly establish a role for promiscuity in the divergence of natural enzymes, we attempted to trace the origins of a bacterial phosphotriesterase ( PTE), an enzyme thought to have evolved for the purpose of degradation of a synthetic insecticide introduced in the 20th century. We surmised that PTE's promiscuous lactonase activity may be a vestige of its progenitor and tested homologues annotated as "putative PTEs" for lactonase and phosphotriesterase activity. We identified three genes that define a new group of microbial lactonases dubbed PTE-like lactonases ( PLLs). These enzymes proficiently hydrolyze various lactones, and in particular quorum-sensing N-acyl homoserine lactones ( AHLs), and exhibit much lower promiscuous phosphotriesterase activities. PLLs share key sequence and active site features with PTE and differ primarily by an insertion in one surface loop. Given their biochemical and biological function, PLLs are likely to have existed for many millions of years. PTE could have therefore evolved from a member of the PLL family while utilizing its latent promiscuous paraoxonase activity as an essential starting point.
Serum paraoxonase (PON1) is a HDL-associated enzyme exhibiting potentially antiatherogenic properties. Here, we examined the common PON1-192R/Q human polymorphism. Despite numerous studies, the effect of this polymorphism on the antiatherogenic potential of PON1 is yet unresolved. Our structural model suggests that amino acid 192 constitutes part of the HDL-anchoring surface and active site of PON1. Based on our findings that PON1 is an interfacially activated lipolactonase that selectively binds HDL carrying apolipoprotein A-I (apoA-I) and is thereby greatly stabilized and catalytically activated, we examined the interaction of the PON1-192 isozymes with reconstituted HDL-apoA-I particles. We found that PON1 position 192 is indeed involved in HDL binding. The PON1-192Q binds HDL with a 3-fold lower affinity than the R isozyme and consequently exhibits significantly reduced stability, lipolactonase activity, and macrophage cholesterol efflux. We also observed the lower affinity and stability of the 192Q versus the 192R isozyme in sera of individuals belonging to the corresponding genotypes. The observed differences in the properties of PON1-192R/Q isozymes provide a basis for further analysis of the contribution of the 192R/Q polymorphism to the susceptibility to atherosclerosis, although other factors, such as the overall levels of PON1, may play a more significant role.
The past few years have seen significant advances in research related to the 'latent skills' of enzymes-namely, their capacity to promiscuously catalyze reactions other than the ones they evolved for. These advances regard (i) the mechanism of catalytic promiscuity-how enzymes, that generally exert exquisite specificity, promiscuously catalyze other, and sometimes barely related, reactions; (ii) the evolvability of promiscuous functions-namely, how latent activities evolve further, and in particular, how promiscuous activities can firstly evolve without severely compromising the original activity. These findings have interesting implications on our understanding of how new enzymes evolve. They support the key role of catalytic promiscuity in the natural history of enzymes, and suggest that today's enzymes diverged from ancestral proteins catalyzing a whole range of activities at low levels, to create families and superfamilies of potent and highly specialized enzymes.
Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (> 10(10) per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.
The efficient amplification of genomic libraries, cDNA libraries and other complex mixtures of genes by PCR is impeded by two phenomena: firstly, short fragments tend to be amplified in preference to larger ones; and, secondly, artifactual fragments are generated by recombination between homologous regions of DNA(1). Recombination in this case occurs when a primer is partially extended on one template during one cycle of PCR and further extended on another template during a later cycle. Thus, chimeric molecules are generated, the short ones of which are then preferentially amplified as described in Figure 1. A variety of PCR protocols have been proposed to minimize these problems, most of which rely on high template concentrations and low numbers of PCR cycles(2,3). Clearly, however, such an approach is not viable if little template DNA is available. Here we describe a protocol for amplifying complex DNA mixtures, based on the compartmentalization of genes in a water-in-oil (w/o) emulsion. Template fragments are segregated in the minute aqueous droplets of the emulsion and amplified by PCR in isolation (Fig. 1). This approach alleviates the problems described above while enabling the use of small amounts of template DNA and high numbers of PCR cycles. Box 1 describes an alternative method for generating very stable emulsions for emulsion PCR using the surfactant ABIL EM 90 (Fig. 2).
The goal of in vitro compartmentalization (IVC) is to divide a large reaction between many microscopic compartments(1). This technique was first developed to generate 'artificial cells' for the directed evolution of proteins (Fig. 1). Typically, an aqueous solution of genes and an in vitro transcription-translation system is stirred (or homogenized) into an oil-surfactant mixture to create a water-in-oil (w/o) emulsion with similar to 10(10) aqueous droplets per ml of emulsion. The majority of droplets contain no more than a single gene along with all of the molecular machinery needed to express that gene. The expressed proteins and the products of their catalytic activities cannot leave the droplets, and so genotype is coupled to phenotype in vitro, making it possible to select very large libraries of genes (10(8)-10(11) genes). We describe the advantages and applications of IVC in Box 1. Here we present a protocol for performing a directed evolution experiment by IVC that makes use of one or more w/o emulsions. This procedure involves the generation of a gene library, the performance of a selection, and the subsequent recovery of the selected genes by PCR. We also describe two procedures for converting w/o emulsions to water-in-oil-in- water (w/o/w) emulsions for high-throughput screening using a fluorescence-activated cell sorter (FACS; Box 2 and Fig. 2, and Box 3). Finally, we describe two methods for delivering substrates, regulators and other compounds to the preformed aqueous droplets of a w/o emulsion (Box 4).
Keywords: Biochemistry & Molecular Biology
Homocysteine thiolactone (tHcy) is deemed a risk factor for cardiovascular diseases and strokes, presumably because it acylates the side chain of protein lysine residues ("N-homocysteinylation"), thereby causing protein damage and autoimmune responses. We analysed the kinetics of hydrolysis and aminolysis of tHcy and two related thiolactones (gamma-thiobutyrolactone and N-trimethyl-tHcy), and we have thereby described the first detailed mechanism of thiolactone aminolysis. As opposed to the previously studied (thio and oxo)esters and (oxo)lactones, aminolysis of thiolactones was found to be first order with respect to amine concentration. Anchimeric assistance by the alpha-amino group of tHcy (through general acid/base catalysis) could not be detected, and the Bronsted plot (nucleophilicity versus pK(a)) for aminolysis yielded a slope (beta(nuc)) value of 0.66. These data support a mechanism of aminolysis where the rate-determining step is the formation of a zwitterionic tetrahedral intermediate. The beta(nuc) value and steric factors dictate a regime whereby, at physiological pH values (pH 7.4), maximal reactivity of tHcy is exhibited with primary amine groups with a pKa value of 7.7; this allows the reactivity of various protein amino groups towards N-homocysteinylation to be predicted.
We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was similar to 10-fold faster with PONI mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wildtype. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PONI is less active toward the more toxic optical isomers.
High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL(rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by similar to 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.
Serum paraoxonases (PONs) are calcium-dependent lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the lactonase mechanism of mammalian PON1, a high density lipoprotein-associated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the lactonase mechanism of PON1 and PON3. The pH-rate profile determined for the lactonase activity of PON1 indicated an apparent pK(a) of similar to 7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His(115), His(134), His(184), His(285), Asp(183), and Asp(269)). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the lactonase activity of PONs. The results indicate that the lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His(115)-His(134) dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad.
Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzymecoding genes. We demonstrate the directed evolution of new enzyme variants by screening > 10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying > 10(4) enzyme molecules, in a volume of <10 ferntoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.
The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)(8) barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP). Of particular interest are PTE, which is thought to have evolved within the last several decades, and PHP, an amidohydrolase superfamily member of unknown function, and the closest known homologue of PTE. We found a diverse and partially overlapping pattern of promiscuous activities in these enzymes, including a significant lactonase activity in PTE, esterase activities in both PTE and PHP, and a weak PTE activity in DHO. Directed evolution was applied to improve the promiscuous esterase activities of PTE and PHP. Remarkably, the most recurrent mutation increasing esterase activity in PTE, or PHP, maps to the same location in their superposed 3D structures. The evolved variants also exhibit newly acquired promiscuous activities that were not selected for, including very weak, yet measurable, paraoxonase activity in PHP. Our results illustrate the mechanistic, structural, and evolutionary links between these enzymes, and highlight the importance of studying laboratory evolution intermediates that might resemble node intermediates along the evolutionary pathways leading to the divergence of enzyme superfamilies.
induced fit is a predominant phenomenon in protein-ligand interactions, yet it is invariably attributed without establishing the existence, let alone the structure, of the initial, low-affinity encounter complex. We determined the crystal structure of the encounter complex on the pathway of ligand binding by IgE antibody SPE7. We show that this complex is formed by a wide range of ligands that initially bind with identical affinity. Nonspecific ligands rapidly dissociate, whereupon the antibody isomerizes to a nonbinding isomer. Specific ligand complexes, however, slowly isomerize to give a high-affinity complex. This isomerization involves backbone and side-chain rearrangements of up to 14 angstrom and the formation of specific hydrogen bonds. The postbinding conformational switch, combined with the prebinding isomerization to an energetically favorable nonbinding isomer, results in a "kinetic discrimination" mechanism that mediates selective binding, by a factor of > 10(3), between highly related ligands that initially bind with the same affinity. This model may apply to proteins that bind multiple ligands in a specific manner or other proteins that, although capable of binding many ligands, are activated by only a few.
Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K-d 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K-d = 1.2 x 10(-7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by
A promiscuous activity of an existing enzyme can confer an evolutionary advantage by providing an immediate response to a new selection pressure and a starting point for the divergence of a new enzyme. This work seeks to examine how this process might take place. Human carbonic anhydrase II (hCAII) is an enzyme that evolved to catalyze the reversible hydration of CO2 and performs this task at a remarkable rate (k(cat) approximate to 10(6) s(-1)). hCAII also exhibits promiscuous activity toward highly activated esters such as 4-nitrophenyl acetate. We describe a much weaker esterase activity of hCAII toward the bulkier and much less activated ester substrate 2-naphthyl acetate (2NA). Directed evolution of hCAII produced a variant with 40-fold higher rates toward 2NA, owing to two mutations: one within the active site (Ala65Val) and one at its mouth (Thr200Ala). Structure-activity studies suggest that these mutations led to adaptation of the active site for bulkier substrates and for the catalysis of nonactivated esters. The mutations did not, however, significantly alter the native activity of hCAII. Our results support the notion that the evolution of a new function can be driven by mutations that increase a promiscuous function (which serves as the starting point for the evolutionary process) but do not harm the native function.
PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals (including humans) and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. PON1 resides on HDL (the "good cholesterol") and is also involved in the prevention of atherosclerosis. Despite this wealth of activities, the identity of PON1's native substrate, namely, the substrate for which this enzyme and other enzymes from the PON family evolved, remains unknown. To elucidate the substrate preference and other,details of PON1 mechanism of catalysis, structure-activity studies were performed with three groups of substrates that are known to be hydrolyzed by PON1: phosphotriesters, esters, and lactones. We found that the hydrolysis of aryl esters is governed primarily by steric factors and not the pK(a) of the leaving group. The rates of hydrolysis of aliphatic esters are much slower and show a similar dependence on the pK(n) of the leaving group to that of the nonenzymatic reactions in solution, while the aryl phosphotriesters show much higher dependence than the respective nonenzymatic reaction. PON1-catalyzed lactone hydrolysis shows almost no dependence on the pK(a) of the leaving group, and unlike all other substrates, lactones seem to differ in their K-M rather than k(cat) values. These, and the relatively high rates measured with several lactone substrates (k(cat)/K-M approximate to 10(6) M-1 s(-1)) imply that PON1 is in fact a lactonase.
The availability of vast gene repertoires from both natural sources (genomic and cDNA libraries) and artificial sources (gene libraries) demands the development and application of novel technologies that enable the screening or selection of large libraries for a variety of enzymatic activities. We describe recent developments in the selection of enzyme-coding genes for directed evolution and functional genomics. We focus on HTS approaches that enable selection from large libraries (> 10(6) gene variants) with relatively humble means (i.e. non-robotic systems), and on in vitro compartmentalization in particular.
In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually 0 1 transcribed and translated. Here, we present a new application of IVC for the selection of DNA-nuclease inhibitors. We developed a nano-droplets delivery system that allows the transport of various solutes, including 0 metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. Thus, genes encoding nuclease inhibitors survived the digestion and were subsequently amplified and isolated. Selection is therefore directly for inhibition, and not for binding of the nuclease. The stringency of selection can be easily modulated to give high enrichments (100-500-fold) and recoveries. We demonstrated its utility by selecting libraries of the gene encoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) evolved inhibitors for inhibition of another colicin (ColE7). The in vitro show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that determine the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. The M vitro evolution process confirms earlier hypotheses regarding the "dual recognition" binding mechanism and the way in which new colicin-immunity pairs diverged from existing ones. (C) 2004 Elsevier Ltd. All rights reserved.
Recent developments have been made in the application of directed evolution to achieve the efficient heterologous expression of proteins in Escherichia coli and yeast by increasing the stability and solubility of the protein in the host environment. One interesting conclusion that emerges is that the evolutionary process often improves the stability and solubility of an intermediate (apoprotein, proprotein or folding intermediate) that otherwise constitutes a bottleneck to functional expression, rather than altering the protein's final state.
How proteins with new functions (e. g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear. This ability is dependent on the induction of new phenotypic traits by a small number of mutations (plasticity). But mutations often have deleterious effects on functions that are essential for survival. How are these seemingly conflicting demands met at the single-protein level? Results from directed laboratory evolution experiments indicate that the evolution of a new function is driven by mutations that have little effect on the native function but large effects on the promiscuous functions that serve as starting point. Thus, an evolving protein can initially acquire increased fitness for a new function without losing its original function. Gene duplication and the divergence of a completely new protein may then follow.
Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.
S-Adenosylmethionine (AdoMet) is a commonly used cofactor, second only to ATP in the variety of reactions in which it participates. It is the methyl donor in the majority of methyl transfer reactions, including methylation of DNA, RNA, proteins and small molecules. Almost all structurally characterised methyltransferases share a conserved AdoMet-dependent methyltransferase fold, in which AdoMet is bound in the same orientation. Although potential interactions between the cofactor and methyltransferases have been inferred from crystal structures, there has not been a systematic study of the contributions of each functional group to binding. To explore the binding interaction we synthesised a series of seven analogues of the methyltransferase inhibitor S-adenosylhomocysteine (AdoHcy), each containing a single modi cation, and tested them for the ability to inhibit methylation by HhaI and HaeIII DNA methyltransferase. Comparison of the K-i values highlights the structural determinants for cofactor binding, and indicates which nucleoside and amino acid functional groups contribute significantly to AdoMet binding. An understanding of the binding of AdoHyc to methyltransferases will greatly assist the design of AdoMet inhibitors.
Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, good cholesterol) and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 Angstrom. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.
We present a new and facile method to evaluate w/o/w emulsions containing fluorescent markers by flow cytometry. Flow cytometry allows simultaneous measurement of w/o/w emulsion droplets "marked" with a fluorescent marker or "blank" without the need for complicated sample preparation. The yield of preparation of the w/o/w emulsion and the release rate of the fluorescent marker FITC-BSA were investigated by this new method. The release fraction (after 24 h) of FITC-BSA from the w/o/w emulsion decreased with increasing concentration of FITC-BSA inside the internal phase, just like the release fraction of NaCl as marker from the w/o/w emulsion. Flow cytometry results show that the yield and release behavior in w/o/w emulsions are in agreement with results reported by more complicated methods.
Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40,000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes. (C) 2003 Elsevier Inc. All rights reserved.
Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON1 variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved POW variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.
Engineering the specificity of DNA-modifying enzymes has proven extremely challenging, as sequence recognition by these enzymes is poorly understood. Here we used directed evolution to generate a variant of HaeIII methyltransferase that efficiently methylates a novel target site. M.HaeIII methylates the internal cytosine of the canonical sequence GGCC, but there is promiscuous methylation of a variety of non-canonical sites, notably AGCC, at a reduced rate. Using in vitro compartmentalization (IVC), libraries of M.HaeIII genes were selected for the ability to efficiently methylate AGCC. A two-step mutagenesis strategy, involving initial randomization of DNA-contacting residues followed by randomization of the loop that lies behind these residues, yielded a mutant with a 670-fold improvement in catalytic efficiency (k(cat)/K-m(DNA)) using AGCC and a preference for AGCC over GGCC. The mutant methylates three sites efficiently (AGCC, CGCC and GGCC). Indeed, it methylates CGCC slightly more efficiently than AGCC. However, the mutant discriminates against other noncanonical sites, including TGCC, as effectively as the wildtype enzyme. This study provides a rare example of a laboratory-evolved enzyme whose catalytic efficiency surpasses that of the wild-type enzyme with the principal substrate.
Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross-reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4-dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross-reactants are themselves bound specifically-close derivatives of these cross-reactants show very low or no binding to SPET It has been suggested that cross-reactivity is simply due to "hydrophobic stickiness", nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross-reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1-8. By comparing the sequences and binding patterns of SPE7 and B1-8, we address the relationship between affinity maturation, specificity, and cross-reactivity.
Complex organisms have evolved from a limited number of primordial genes and proteins. However, the mechanisms by which the earliest proteins evolved and then served as the origin for the present diversity of protein function are unknown. Here, we outline a hypothesis based on the 'new view' of proteins whereby one sequence can adopt multiple structures and functions. We suggest that such conformational diversity could increase the functional diversity of a limited repertoire of sequences and, thereby, facilitate the evolution of new proteins and functions from old ones.
A single antibody was shown to adopt different binding-site conformations and thereby bind unrelated antigens. Analysis by both x-ray crystallography and pre-steady-state kinetics revealed an equilibrium between different preexisting isomers, one of which possessed a promiscuous, low-affinity binding site for aromatic ligands, including the immunizing hapten. A subsequent induced-fit isomerization led to high-affinity complexes with a deep and narrow binding site. A protein antigen identified by repertoire selection made use of an unrelated antibody isomer with a wide, shallow binding site. Conformational diversity, whereby one sequence adopts multiple structures and multiple functions, can increase the effective size of the antibody repertoire but may also lead to autoimmunity and allergy.
We describe the selection of a phosphotriesterase with a very fast k(cat) (over 10(5) s(-1)), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 x 10(7) mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected.
In vitro compartmentalisation in an emulsion was used to physically link proteins to the DNA that encodes them via microbeads. These microbeads can be selected for catalysis, or, as demonstrated here, for binding. Genes encoding a peptide containing an epitope (haemagglutinin) were enriched to near purity from a 10(6)-fold excess of genes encoding a different peptide by two rounds of selection using flow cytometry, indicating similar to 1000-fold enrichment per round. Single beads can be isolated using How sorting and the single gene on the bead amplified by polymerase chain reaction. Hence, the entire process can be performed completely in vitro. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on compartmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5'-GCGC-3'). Crystallog raphy shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target for methylation) and two target recognition loops (loops I and II) from the small domain make almost all the other base-specific interactions. A library of M.HhaI genes was created by randomising all the loop II residues thought to make base-specific interactions and directly determine target specificity. The library was selected for 5'-GCGC-3' methylation. Interestingly, in 11 selected active clones, 10 different sequences were found and none were wild-type. At two of the positions mutated (Ser252 and Tyr254) a number of different amino acids could be tolerated. At the third position, however, all active mutants had a glycine, as in wild-type M.HhaI, suggesting that Gly257 is crucial for DNA recognition and enzyme activity. Our results suggest that recognition of base pairs 3 and 4 of the target site either relies entirely on main chain interactions or that different residues from those identified in the crystal structure contribute to DNA recognition.
The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction-modification systems with novel sequence specificities.
Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an a-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole. (C) 2002 Elsevier Science Ltd. All rights reserved.
It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit sonic activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins-serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities-in the shape of the cavities and the position of catalytic lysine residues-both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active-site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind-inefficient and rudimentary as they are-can provide a considerable selective advantage and a starting point for the evolution of new functions.
Medium effects are normally studied by comparing the rates of reactions in different solvents. However, medium effects at the active site of enzymes differ dramatically from bulk solvents, both in their diversity (the presence of more than one type of "solvent") and in their spatial arrangement. We describe medium effects in a simple catalytic system, obtained by systematic alkylation of a polymeric scaffold bearing amine groups to give synzymes that catalyze the Kemp elimination of benzisoxazoles with remarkable efficiency. Our analysis indicates that catalysis by these synzymes is driven primarily by specific, localized enzyme-like medium effects, and these effects seem to differ dramatically from the nonspecific medium effects (i.e., desolvation activation) exhibited by solvents. Ligand-binding studies indicate that the synzyme active sites provide localized microenvironments affording a combination of hydrophobic and apolar regions on one hand and dipolar, protic, and positively charged on the other. Such localized microenivronments are not available in bulk solvents. A Bronsted (leaving group) analysis indicates that, in comparison to solvent catalysis, the efficiency of synzyme catalysis shows little sensitivity to leaving group pK(a). We show that enzyme-like medium effects alone, in the absence of efficient positioning of the catalytic amine base relative to the substrate, can give rise to rate accelerations as high as 10(5), for both activated and nonactivated substrates. Supported by the accidental identification of active sites on the surfaces of noncatalytic proteins and the promiscuous activities found in many enzymes, our findings suggest that the interfaces of protein surfaces and their hydrophobic cores provide a microenvironment that is intrinsically active and may serve as a basis for further evolutionary improvements to give proficient and selective enzymes.
D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k = 1-7 s(-1)). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k <0.02 s(-1)). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA. (C) 1999 Academic Press.
The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available. In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters. ii group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters. In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis. Levels of expression were dependent on the sequence of V-L used. Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original V-H and V-L regions are required for hapten binding, although the V-H is more crucial for catalysis. By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies. (C) 1998 Elsevier
A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (k(cat); as expected from the higher intrinsic reactivity of Np-esters); however, the rate acceleration (k(cat)/k(uncat)) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate (1/K-M) and therefore k(cat)/K-M are significantly higher. The best example is antibody D2.4 having a k(cat)/K-M value of 64 s(-1). M(-1) with the Nbzl-ester and 9400 s-1 . M(-1) with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl phosphonate TSA versus the Nbzl-ester substrate (K-S/K-TSA or K-M/K-i) correlates well with the observed rate enhancement (k(cat)/k(uncat)). For the Np-ester, however, stabilisation of the transition state (as reflected by K-S/K-TSA and by the catalytic proficiencies, k(cat)/K-M/k(uncat)) does not fully account for the catalytic power (k(cat)/k(uncat)), indicating a more complex catalytic mechanism than simply transition-state stabilization. A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bon
The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.
Upon testing the ability of several strains of mice to elicit esterolytic antibodies after immunization with a p-nitrobenzyl phosphonate hapten, we have found that the occurrence of catalytic antibodies in SJL and MRL/lpr autoimmune mice is dramatically higher than in normal mouse strains (e,g., the wild-type MRL/++ or BALB/c). Fewer than 10 catalytic clones are usually obtained from a single fusion of lymphocytes taken from normal mice, whereas several hundred catalytic clones are obtained in SJL or MRL/lpr mice. Differences in the numbers of hapten-binding clones do not account for the high occurrences of catalytic clones in these strains, This phenomenon prevailed in the early responses; in both SJL and MRL/lpr mice a significant decline in the appearance of catalytic clones was observed after multiple immunizations, Esterolytic antibodies were not found in MRL/lpr mice immunized with haptens that do not mimic the transition state for the hydrolysis of the ester substrate (e,g., with a substrate analog). The catalytic antibodies manifest high specificity to the antigen and variability in their binding and catalytic properties. The use of autoimmunity-prone mice may greatly expand the repertoire of catalytic clones elicited against a transition-state analog hapten. More intriguing is the possible linkage between autoimmunity and the appearance of catalytic antibodies. These results suggest that there is normally a selection against the expression of certain variable genes encoding antibodies with catalytic activity.
Background: Antibodies with catalytic properties can be prepared by eliciting an antibody response against 'transition state analog' haptens. The specificity, rate and number of reaction cycles observed with these antibodies more closely resemble the properties of enzymes than any of the many other known enzyme-mimicking systems. Results: We have determined to 3 angstrom resolution the first X-ray structure of a catalytic antibody Fab. This antibody catalyzes the hydrolysis of a p-nitrophenyl ester. In conjunction with binding studies in solution, this structure of the uncomplexed site suggests a model for transition state fixation where two tyrosines mimic the oxyanion binding hole of serine proteases. A comparison with the structures of known Fabs specific for low molecular weight haptens reveals that this catalytic antibody has an unusually long groove at its combining site. Conclusion: Since transition state analogs contain elements of the desired product, product inhibition is a severe problem in antibody catalysis. The observation of a long groove at the combining site may relate to the ability of this catalytic antibody to achieve multiple cycles of reaction.
Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pa-dependent binding near physiological pH. Three monoclonal antibodies were selectively modified, each under different conditions, with the resultant loss of binding activity at pH > 8 which is recovered at pH <6. Recovery and loss of binding are ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of the resulting 3-nitrotyrosine side chain (pK(a) similar to 7) at the binding site of these antibodies. pH on-off dependency of binding activity, common to all TNM-modified antibodies studied by us so far, may find use in a variety of applications in which controlled modulation under mild conditions is required.
A prerequisite to the design and engineering of catalytic antibodies is the knowledge of their structure and in particular which residues are involved in binding and catalysis. We compared the structure and catalytic properties of a series of six monoclonal antibodies which were all raised against a p-nitrophenyl (PNP) phosphonate and which catalyze the hydrolysis of p-nitrophenyl esters. Three of the antibodies (Group I) have similar light and heavy chain variable regions. The other three antibodies have similar V-L regions of which two (Group II) have V-H regions from the MOPC21 gene family and the remaining one (Group III) a V-H from the MC101 gene family making a total of three different groups based on their V region sequences. The structural division into groups is paralleled by the differences in binding constants to hapten analogs, substrate specificity and the susceptibility of the catalytic activity of the antibodies to chemical modification of tryptophan and arginine residues. The relative binding of a transition state analog to the binding of substrate is much higher for the Group I antibodies than for the other groups. Only the Group I antibodies can catalyze the hydrolysis of a carbonate substrate. However all of the antibodies lose catalytic activity upon specific tyrosine modification which highlights the importance of tyrosine in the active site of the antibodies. Thus, antibodies raised against a single hapten can give antibodies with different structures, and correspondingly different specificities and catalytic properties.
DBU (1,8-diazabicyclo[5.4.0]undecene) was found to efficiently mediate the transesterification of p-nitrophenyl (PNP) phosphonates by various alcohols. The reactions of bis-PNP phosphonates in the presence of DBU, using both primary and secondary alcohols, phenols and amines, proceed rapidly and with high yield to afford the corresponding monoalkyl/aryl mono-PNP phosphonates as sole products (1, Scheme 2). The resulting monoalkyl/aryl mono-PNP phosphonates can be further reacted with a second alcohol to give the corresponding differently disubstituted phosphonates 3, or selectively hydrolysed to yield the monoalkyl/aryl phosphonic acids 2. We have applied this chemistry to the preparation of a series of phosphono ester transition state analogues 11a-e (Scheme 3) that were used as haptens for raising catalytic antibodies.
The low abundance and activity of catalytic antibodies are major obstacles to their selection from the virtually unlimited repertoire of antibody binding sites. The requirement for new screening methodologies is further emphasized by the availability of combinatorial libraries, in which a functional polypeptide has to be selected out of millions of possibilities. We present a simple and sensitive screening approach (termed catELISA) based on immobilized substrates and immunodetection of the end product of the catalyzed reaction. The feasibility of catELISA is demonstrated here by the generation of potent ester-hydrolyzing antibodies by direct screening of hybridoma supernatants. We show that this approach is not only facile but general: it is not limited by type of reaction, substrate, or catalyst (enzymes, catalytic antibodies, chemical catalysts). catELISA opens a route to catalytic antibodies that replaces existing lengthy and arduous methods, thus allowing us to expand their number and improve their quality and to address questions that would otherwise be difficult to answer.
We present here a newly developed approach based on DBU catalyzed transesterification of bis-PNP-phosphonates, used for the preparation of phosphonoesters and their protein conjugates. These are being used, as transition state analogs, to elicit catalytic antibodies having enzyme-like properties.