Publications

Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 β-subunit with attenuation of the complex's three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome's enzymatic subunits but rather propagates through the non-catalytic subunit PSMB4. This way of 20S proteasome-specific attenuation opens avenues for decoupling the 20S and 26S proteasome degradation pathways as well as for developing selective 20S proteasome inhibitors.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

Enzymes are well known for their catalytic abilities, some even reaching \u201ccatalytic perfection\u201d in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate-handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a \u201csubstrate-handle-binding-first\u201d mechanism (\u201cbinding-first\u201d for brevity) instead of \u201cchemistry-first\u201d and we are, therefore, left to wonder what the role of non-catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back-to-back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as \u201cuniform binding\u201d (parallel stabilization of all enzyme-bound states to the same degree). Indeed, we show that a uniform-binding proto-catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate-handle-binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of \u201cbinding-first\u201d enzyme families like the aldolase superfamily.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/ RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple βα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one β-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Natural product biosynthesis (NPB) is the Panda's Thumb of evolutionary biochemistry. Arm races between organisms, and ever-changing environments, result in relentless innovation. This review focusses on enzyme evolution in NPB. First, we review cases of de novo emergence, whereby a completely new enzymatic activity arose in a ligand-binding protein, or a new enzyme emerged including a completely new scaffold. Second, we briefly review the current models for enzyme evolution, and how they explain the inherent promiscuity of NPB enzymes and their tendency to produce multiple related products. We thus suggest that NPB enzymes a priori evolved to generate a specific product; they are, however, trapped in a multifunctional, generalist evolutionary state and thereby produce a diversity of products.

Oligomeric proteins are central to life. Duplication and divergence of their genes is a key evolutionary driver, also because duplications can yield very different outcomes. Given a homomeric ancestor, duplication can yield two paralogs that form two distinct homomeric complexes, or a heteromeric complex comprising both paralogs. Alternatively, one paralog remains a homomer while the other acquires a new partner. However, so far, conflicting trends have been noted with respect to which fate dominates, primarily because different methods and criteria are being used to assign the interaction status of paralogs. Here, we systematically analyzed all Saccharomyces cerevisiae and Escherichia coli oligomeric complexes that include paralogous proteins. We found that the proportions of homo-hetero duplication fates strongly depend on a variety of factors, yet that nonetheless, rigorous filtering gives a consistent picture. In E. coli about 50%, of the paralogous pairs appear to have retained the ancestral homomeric interaction, whereas in S. cerevisiae only ~10% retained a homomeric state. This difference was also observed when unique complexes were counted instead of paralogous gene pairs. We further show that this difference is accounted for by multiple cases of heteromeric yeast complexes that share common ancestry with homomeric bacterial complexes. Our analysis settles contradicting trends and conflicting previous analyses, and provides a systematic and rigorous pipeline for delineating the fate of duplicated oligomers in any organism for which protein-protein interaction data are available.

Traditionally, enzymes are referred to as remarkably fast and specific catalysts. The fact that many enzymes are capable of catalyzing other reactions, besides the one they physiologically specialize in, or evolved for, is definitely not new. Since a long time, breaches of specificity, or promiscuity, of enzymes have been recognized. Early examples of enzyme promiscuity include pyruvate decarboxylase,¹ carbonic anhydrase,² pepsin,³ chymotrypsin,⁴ and L-asparaginase.⁵ However, these early discussions of enzymatic versatility were scarce, and until recently, the wider implications of this darker side of enzymes were largely ignored. During the last decade, protein, and especially enzyme promiscuity, received considerable attention, and their importance in various contexts was systematically studied. Reviews by OBrien and Herschlag,⁶ and later Copley,⁷ were the first to highlight the mechanistic and evolutionary implications of promiscuity. Other, more recent reviews have focused on the practical implications of promiscuity in organic synthesis,^1,8,9 on promiscuity and divergence in certain enzyme families,¹⁰¹³ on mechanistic aspects of promiscuity,^1,14 and on promiscuity in the context of protein evolution¹⁴ and design.¹⁵ The primary focus of this chapter is the role of promiscuity in the evolution of new enzyme functions. New enzymes have constantly emerged throughout the natural history of this planet. Over the past decades, enzymes that degrade synthetic chemicals were introduced to the biosystem,¹⁶²⁰ and enzymes associated with drug resistance,²¹²⁴ provide vivid examples of how rapid the evolution of new enzymatic functions can be. The first direct connection between protein evolution and promiscuity was made in 1976 by Jensen.

This Special Issue is composed of 10 reviews that delve into the intricacies behind enzyme promiscuity and evolution, an area that is of increasing interest in the biological research community. In particular, the reviews in this Special Issue explore enzyme promiscuity and evolution in the context of cellular metabolism, as discussed in this introductory Editorial. It is our hope that you enjoy these fascinating and informative reviews and we wish to thank the authors for their compelling contributions to The FEBS Journal. 

The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristically in αβα sandwich enzyme domains, N-helix binding sites dominate, where the phosphate moiety sits atop the N terminus of an α-helix. This discrepancy is explained by the observation that N-helix binding is uniquely realized by short, contiguous sequences with reduced amino acid diversity, foremost Gly, Ser, and Thr. The latter two amino acids preferentially interact with both the backbone amide and the side-chain hydroxyl (bidentate interaction) to promote binding by short sequences. We conclude that the first αβα sandwich domains emerged from shorter and simpler polypeptides that bound phospho-ligands via N-helix sites.

Chalcone isomerases are plant enzymes that perform enantioselective oxa-Michael cyclizations of 2'-hydroxychalcones into flavanones. An X-ray crystal structure of an enzyme-product complex combined with molecular dynamics simulations reveal an enzyme mechanism wherein the guanidinium ion of a conserved arginine positions the nucleophilic phenoxide and activates the electrophilic enone for cyclization through Bronsted and Lewis acid interactions. The reaction terminates by asymmetric protonation of the carbanion intermediate syn to the guanidinium. Interestingly, bifunctional guanidine- and urea-based chemical reagents, increasingly used for asymmetric organocatalytic applications, share mechanistic similarities with this natural system. Comparative protein crystal structures and molecular dynamics simulations further demonstrate how two active site water molecules coordinate a hydrogen bond network that enables expanded substrate reactivity for 6'-deoxychalcones in more recently evolved type-2 chalcone isomerases.

How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of Bacillus subtilis glutamate dehydrogenase mutants across 19 different environments-from phenotypically homogeneous single-cell populations in liquid media to heterogeneous biofilms, plant roots and soil populations. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. However, 84% of the tested alleles showed opposing fitness effects under different growth conditions (sign environmental pleiotropy). In colony biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell populations the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred during biofilm development. Overall, for each condition, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated as a result of the combined action of selection, pleiotropy and chance. However, over longer periods and in multiple environments, nearly all of this diversity would be lost-across all the environments and conditions that we tested, the wild type was the fittest allele.

Protein Science, 27: 12191226. doi:10.1002/pro.2928 Equation 1 has been erroneously printed, and should read as follows: (Formula presented.) whereby f is the fraction of an allele competing against wild-type, f
₀ is the fraction of this allele at generation zero, s is its selection coefficient, and n is the number of generations. Note that if s > 0, as n increases, f/f
₀ increases exponentially; however, given a finite population size, this value will plateau at fixation (i.e., complete takeover of the competing allele). Similarly, if s

Chemists, including the most prominent ones, have always had a profound interest in living systems and biomolecules. Here, I describe few historical endeavors at the interface of Chemistry and Biology that inspired me and shaped my research interests. These examples highlight the uniqueness of implementing a chemist's mindset to fundamental biological questions, by obtaining biological insight via structure-function analyses and by implementing the fundamentals of physical chemistry. I therefore see Chemical Biology as the study of biological systems and molecules with the mindset of a chemist, and in my case, of a physical organic chemist. To me, addressing biological questions with the toolset and mindset of a chemist seems so obvious and natural that there may be no need to define a specialized sub-discipline.

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase ( Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic-stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco's maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD(+), thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbonconserving photorespiration pathway.

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.

How individual enzymes evolved is relatively well understood. However, individual enzymes rarely confer a physiological advantage on their own. Judging by its current state, the emergence of metabolism seemingly demanded the simultaneous emergence of many enzymes. Indeed, how multicomponent interlocked systems, like metabolic pathways, evolved is largely an open question. This complexity can be unlocked if we assume that survival of the fittest applies not only to genes and enzymes but also to the metabolites they produce. This review develops our current knowledge of enzyme evolution into a wider hypothesis of pathway and network evolution. We describe the current models for pathway evolution and offer an integrative metabolite-enzyme coevolution hypothesis. Our hypothesis addresses the origins of new metabolites and of new enzymes and the order of their recruitment. We aim to not only survey established knowledge but also present open questions and potential ways of addressing them.

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.

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.

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.

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.

DNA methyltransferases (MTases) constitute an attractive target for protein engineering, thus opening the road to new ways of manipulating DNA in a unique and selective manner. Here, we review various aspects of MTase engineering, both methodological and conceptual, and also discuss future directions and challenges. Bacterial MTases that are part of restriction/modification (R/M) systems offer a convenient way for the selection of large gene libraries, both in vivo and in vitro. We review these selection methods, their strengths and weaknesses, and also the prospects for new selection approaches that will enable the directed evolution of mammalian DNA methyltransferases (Dnmts). We explore various properties of MTases that may be subject to engineering. These include engineering for higher stability and soluble expression (MTases, including bacterial ones, are prone to misfolding), engineering of the DNA target specificity, and engineering for the usage of S-adenosyl-L-methionine (AdoMet) analogs. Directed evolution of bacterial MTases also offers insights into how these enzymes readily evolve in nature, thus yielding MTases with a huge spectrum of DNA target specificities. Engineering for alternative cofactors, on the other hand, enables modification of DNA with various groups other than methyl and thus can be employed to map and redirect DNA epigenetic modifications.

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 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.

When asked to edit an issue of Current Opinion in Chemical Biology focused on biocatalysis, we felt that it was high time to cover the esoteric namely, the enzymatic reactions leading to biosynthesis of natural products in ecologically diverse niches and/or in somewhat unusual organisms. High time not because we have exhausted the study of standard enzymes, but because specialized enzymology is revealing new and beautiful aspects of Nature's chemistry, and because several of these niches are actually quite central with respect to life on this planet.

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.

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.

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

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.

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.

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.

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.

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.

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 Ca^{2 +}. 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 Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-Å upward displacement towards the enzyme's surface of the catalytic Ca^{2 +} 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 Ca^{2 +} 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.

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.

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.

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.

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 ≤5 × 10⁴ M ^-1s^-1). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k_cat/K_m, and by catalyzing the elimination of non-activated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher kcat values. The best KE59 variants exhibited k_cat/K_m values up to 0.6 × 10⁶ M^-1s^-1, and k_cat/k _uncat values of ≤10⁷ almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.

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 ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 10 ⁷ M ^-1 min ^-1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) -4 in favor of the R _P isomer of a cyclosarin analog in wild-type PON1, to E > 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.

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.

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.

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.

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.

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.

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 ∼100 amino acid fragments derived from tachylectin-2 - a monomeric, 236 amino acids, five-bladed β-propeller. Their structures reveal a unique pentameric assembly and novel β-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 β-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.

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.

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.

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.

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.

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 10⁵ 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 ^-1s^-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.

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.

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).

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.

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 reconstitutedHDL(rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by ∼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 withWTPON1, 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 amodel in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.

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.

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.

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_a 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 ≈ 10⁶ M^-1 s^-1) imply that PON1 is in fact a lactonase.

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.

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 Å. PON1 is a six-bladed β-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.

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.

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.

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 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.

Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pH-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 a∼ 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