Publications

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Enzymes are well known for their catalytic abilities, some even reaching “catalytic perfection” 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 “substrate‐handle‐binding‐first” mechanism (“binding‐first” for brevity) instead of “chemistry‐first” 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 “uniform binding” (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 “binding‐first” enzyme families like the aldolase superfamily.

Phytoplankton are key components of the oceanic carbon and sulfur cycles1. During bloom events, some species can emit large amounts of the organosulfur volatile dimethyl sulfide (DMS) into the ocean and consequently the atmosphere, where it can modulate aerosol formation and affect climate2,3. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic interactions. Yet, its role in microbial predator–prey interactions remains elusive with contradicting evidence for its role in either algal chemical defence or in the chemo-attraction of grazers to prey cells4,5. Here we investigated the signalling role of DMS during zooplankton–algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme dimethylsulfoniopropionate lyase (DL) in the bloom-forming alga Emiliania huxleyi6. We inhibited DL activity in E. huxleyi cells in vivo using the selective DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate7 and overexpressed the DL-encoding gene in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as an anti-grazing chemical defence but paradoxically enhanced predation by the grazer Oxyrrhis marina and other microzooplankton and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina growth. Overall, our results demonstrate that DMS-mediated grazing may be ecologically important and prevalent during prey–predator dynamics in aquatic ecosystems. The role of algal DMS revealed here, acting as an eat-me signal for grazers, raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean.

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.

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. 

Aminoacyl-tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2–3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 10
5-fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H-bonding with polar active site groups. This ‘hydroxyl paradox’ is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post-transfer editing – hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates (‘negative selection’).

As soon as I began planning a volume on enzyme engineering and evolution for Methods in Enzymology, I faced a dilemma. This topic is broad, encompassing multiple fields and approaches, computational as well as experimental ones, and is pursued by many research groups. By the time I finalized a minimal list of topics and contributors that I deemed central to this area, I had two volumes in hand: one focusing on engineering methods of general applicability (Volume 643) and the other focusing on the engineering of specific enzymes (Volume 644).

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: 1219–1226. 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.

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.

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.

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substratebinding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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

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.

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.

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.

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.

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 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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.