Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) ...effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3–6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.
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•FuncLib is a new method that designs diverse multipoint mutants in enzyme active sites•Designs are efficient and functionally diverse, bypassing high-throughput screening•Designs exhibit up to 4 orders of magnitude improvement in several activities•FuncLib is implemented as a web-server (http://funclib.weizmann.ac.il)
Khersonsky et al. present FuncLib, an automated method for designing catalytic repertoires using phylogenetic analysis and Rosetta design calculations. FuncLib resulted in efficient enzymes, including new hydrolases with the potential to treat nerve agent poisoning.
Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a ...typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
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•A new computational method is used to stabilize five recalcitrant proteins•Designed variants show higher expression and stability with unmodified function•A designed human acetylcholinesterase variant expresses solubly in bacteria•The method is fully automated and implemented on a webserver
Heterologous expression of proteins and their mutants often results in misfolding and aggregation. Goldenzweig et al. (2016) developed an automated algorithm for protein stabilization requiring minimal experimental testing; for instance, the five tested variants of human acetylcholinesterase showed ≥100-fold higher soluble bacterial expression and higher melting temperatures than wild-type.
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 ∼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) ∼ 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.
The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of ...metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
Abstract
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 (kcat/KM) of 5 × 106 M−1 min−1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 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 kcat/KM ≥ 5 × 107 M−1 min−1 and Russian VX (RVX) with a kcat/KM ≥ 107 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 kcat/KM > 5 × 107 M−1 min−1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.
Phenotypic mutations (errors occurring during protein synthesis) are orders of magnitude more frequent than genetic mutations. Consequently, the sequences of individual protein molecules transcribed ...and translated from the same gene can differ. To test the effects of such mutations, we established a bacterial system in which an antibiotic resistance gene (TEM-1 β-lactamase) was transcribed by either a high-fidelity RNA polymerase or its error-prone mutant. This setup enabled the analysis of individual mRNA transcripts that were synthesized under normal or error-prone conditions. We found that an increase of ≈20-fold in the frequency of transcription errors promoted the evolution of higher TEM-1 expression levels and of more stable enzyme variants. The stabilized variants exhibited a distinct advantage under error-prone transcription, although under normal transcription they conferred resistance similar to wild-type TEM-1. They did so, primarily, by increasing TEM-1's tolerance to destabilizing deleterious mutations that arise from transcriptional errors. The stabilized TEM-1 variants also showed increased tolerance to genetic mutations. Thus, although phenotypic mutations are not individually subjected to inheritance and natural selection, as are genetic mutations, they collectively exert a direct and immediate effect on protein fitness. They may therefore play a role in shaping protein traits such as expression levels, stability, and tolerance to genetic mutations.
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.
•Directed evolution employed to create an efficient paraoxon hydrolyzing PON1.•Effective post-exposure treatment of acute paraoxon intoxication demonstrated.•Novel bioscavenger can be used as a stand-alone or combined antidotal treatment.•A dual PON1+rGOT1 treatment may improve survival and recovery post intoxication.
Recent years have seen an increasing number of incidence, in which organophosphate nerve agents (OPNAs) have been used against civilians with devastating outcomes. Current medical countermeasures ...against OPNA intoxications are aimed at mitigating their symptoms, but are unable to effectively prevent them. In addition, they may fail to prevent the onset of a cholinergic crisis in the brain and its secondary toxic manifestations. The need for improved medical countermeasures has led to the development of bioscavengers; proteins and enzymes that may prevent intoxication by binding and inactivating OPNAs before they can reach their target organs. Non-catalytic bioscavengers such as butyrylcholinesterase, can rapidly bind OPNA molecules in a stoichiometric and irreversible manner, but require the administration of large protein doses to prevent intoxication. Thus, many efforts have been made to develop catalytic bioscavengers that could rapidly detoxify OPNAs without being inactivated in the process. Such enzymes may provide effective prophylactic protection and improve post-exposure treatments using much lower protein doses. Here we review attempts to develop catalytic bioscavengers using molecular biology, directed evolution and enzyme engineering techniques; and natural or computationally designed enzymes. These include both stoichiometric scavengers and enzymes that can hydrolyze OPNAs with low catalytic efficiencies. We discuss the catalytic parameters of evolved and engineered enzymes and the results of in-vivo protection and post-exposure experiments performed using OPNAs and bioscavengers. Finally, we briefly address some of the challenges that need to be met in order to transition these enzymes into clinically approved drugs.
•Pre-treatment with catalytic bioscavengers can prevent nerve agent intoxications.•They may also be effectively used for post-exposure therapy in certain cases.•Obtained from natural enzymes using directed evolution and computational design.•Clinical application requires research of safety, stability, delivery and production.
•Optimization plateaus are common when engineering enzymes for higher catalytic efficiency.•These plateaus relate to fundamental properties of evolutionary fitness landscapes.•Marginal protein ...stability is a common cause of plateauing that can be easily overcome.•Activity tradeoffs and epistatic effects are other causes of optimization plateaus.
The practical need for highly efficient enzymes presents new challenges in enzyme engineering, in particular, the need to improve catalytic turnover (kcat) or efficiency (kcat/KM) by several orders of magnitude. However, optimizing catalysis demands navigation through complex and rugged fitness landscapes, with optimization trajectories often leading to strong diminishing returns and dead-ends. When no further improvements are observed in library screens or selections, it remains unclear whether the maximal catalytic efficiency of the enzyme (the catalytic ‘fitness peak’) has been reached; or perhaps, an alternative combination of mutations exists that could yield additional improvements. Here, we discuss fundamental aspects of the process of catalytic optimization, and offer practical solutions with respect to overcoming optimization plateaus.
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