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Analysis of the structures and dynamics of intermediates and engineered mutants from directed protein evolution experiments reveals how dynamic conformational changes are harnessed across evolutionary trajectories to generate new catalytic functions. Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.

Enzymes can exist in multiple stable functional states or sub-states, revealing substantial molecule-to-molecule heterogeneity in catalytic rates even within populations expressed from a clonal gene. Increasing evidence suggests that biological systems have evolved to exploit these functional sub-states to regulate reaction rates and enzyme multi-functionality or promiscuity. Thus, understanding such heterogeneity in evolutionary dynamics is central to elucidating the mechanisms underlying enzyme adaptability and evolvability. To address this, we analyze the evolutionary changes in functional sub-states using single-molecule kinetics. We measure the functional sub-states of wild-type phosphotriesterase (PTE) and 18 evolved variants spanning a trajectory from native PTE activity to promiscuous arylesterase function. This is further supported by an investigation into the conformational sub-states of selected variants using molecular dynamics simulations. Our results reveal that functional optimization is tightly coupled with the redistribution of underlying functional and conformational sub-states, particularly involving the open and closed dynamics of functionally essential loop 7. These findings provide direct evidence that enzyme evolution involves coordinated shifts in conformational heterogeneity and its functional manifestations, together shaping enzyme function. This study uses single-molecule approaches to detect changes in functional sub-states during enzyme evolution and highlights that coordinated shifts in conformational heterogeneity, and the underlying functional sub-states are key to enzyme function.

Automated design of enzymes with wild-type-like catalytic properties has been a long-standing but elusive goal. Here, we present a general, automated method for enzyme design through combinatorial backbone assembly. Starting from a set of homologous yet structurally diverse enzyme structures, the method assembles new backbone combinations and uses Rosetta to optimize the amino acid sequence, while conserving key catalytic residues. We apply this method to two unrelated enzyme families with TIM-barrel folds, glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase-like lactonases (PLLs), designing 43 and 34 proteins, respectively. Twenty-one GH10 and seven PLL designs are active, including designs derived from templates with <25% sequence identity. Moreover, four designs are as active as natural enzymes in these families. Atomic accuracy in a high-activity GH10 design is further confirmed by crystallographic analysis. Thus, combinatorial-backbone assembly and design may be used to generate stable, active, and structurally diverse enzymes with altered selectivity or activity. Computationally designed enzymes often show lower activity or stability than their natural counterparts. Here, the authors present an evolution-inspired method for automated enzyme design, creating stable enzymes with accurate active site architectures and wild-type-like activities.

Characterization of three DUF89 subfamilies representing all domains of life reveals that members function as metal-dependent phosphatases that help eliminate a wide range of damaged phosphometabolites. DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S -sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast ( Saccharomyces cerevisiae ) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.

The microenvironment of the porous channels in enzyme immobilization carriers critically determines the catalytic performance of immobilized enzymes. In this study, we systematically tuned the hydrophobicity/hydrophilicity of the channel microenvironment of hydrogen-bonded organic frameworks (HOFs) by introducing four different substituents (-CH3, -Cl, -F, -NH2) at the 2-position of the phenyl ring of the HOF-101 monomer. These HOF-101 derivatives, which are isostructural to the parent HOF-101, were used to immobilize phosphotriesterase (PTE). The enzyme loading efficiencies ranged from 64.7% to 70.7%, indicating that the substituents had little effect on PTE binding, which primarily relies on carboxyl-residue interactions. Kinetic studies revealed that the hydrophilic -NH2-functionalized HOF-101 (PTE@HOF-101-NH2) exhibited the highest catalytic efficiency (1.43 × 108 M−1 s−1), 2.27 times that of free PTE, while the hydrophobic -CH3 analogue showed reduced activity. Notably, PTE@HOF-101-F demonstrated superior acid resistance (70% relative activity at pH 2) and long-term thermal stability (70% activity retention after 6 h at 70 °C), outperforming other derivatives. In contrast, PTE@HOF-101-NH2 showed the highest activity under mild conditions but suffered from framework dissolution under prolonged harsh treatments. This work demonstrates that fine-tuning the HOF channel microenvironment is an effective strategy to enhance enzyme activity and stability, providing a platform for designing advanced immobilized enzyme systems.

The redesign of enzyme active sites to alter their function or specificity is a difficult yet appealing challenge. Here we used a structure-based design approach to engineer the lactonase SsoPox from Sulfolobus solfataricus into a phosphotriesterase. The five best variants were characterized and their structure was solved. The most active variant, alpha sD6 (V27A-Y97W-L228M-W263M) demonstrates a large increase in catalytic efficiencies over the wild-type enzyme, with increases of 2,210-fold, 163-fold, 58-fold, 16-fold against methyl-parathion, malathion, ethyl-paraoxon, and methyl-paraoxon, respectively. Interestingly, the best mutants are also capable of degrading fensulfothion, which is reported to be an inhibitor for the wild-type enzyme, as well as others that are not substrates of the starting template or previously reported W263 mutants. The broad specificity of these engineered variants makes them promising candidates for the bioremediation of organophosphorus compounds. Analysis of their structures reveals that the increase in activity mainly occurs through the destabilization of the active site loop involved in substrate binding, and it has been observed that the level of disorder correlates with the width of the enzyme specificity spectrum. This finding supports the idea that active site conformational flexibility is essential to the acquisition of broader substrate specificity.

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency ( k cat / K M  = 21 to 38 × 10 6  M −1  min −1 ) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD 50 ) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg −1 i.v.: PTE-2 dosed at 1.3 mg kg −1 i.v. (PTE-2.1) and 2.6 mg kg −1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg −1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier--either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies \"open\" and \"closed\" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with \"evolvability\" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.

The organophosphate (OP)‐hydrolyzing enzyme phosphotriesterase (PTE, variant L7ep‐3a) immobilized within a partially oxidized mesoporous silicon nanoparticle cage is synthesized and the catalytic performance of the enzyme@nanoparticle construct for hydrolysis of a simulant, dimethyl p‐nitrophenyl phosphate (DMNP), and the live nerve agent VX is benchmarked against the free enzyme. In a neutral aqueous buffer, the optimized construct shows a ≈2‐fold increase in the rate of DMNP turnover relative to the free enzyme. Enzyme@nanoparticles with more hydrophobic surface chemistry in the interior of the pores show lower catalytic activity, suggesting the importance of hydration of the pore interior on performance. The enzyme@nanoparticle construct is readily separated from the neutralized agent; the nanoparticle is found to retain DMNP hydrolysis activity through seven decontamination/recovery cycles. The nanoparticle cage stabilizes the enzyme against thermal denaturing and enzymatic (trypsin) degradation conditions relative to free enzyme. When incorporated into a topical gel formulation, the PTE‐loaded nanoparticles show high activity toward the nerve agent VX in an ex vivo rabbit skin model. In vitro acetylcholinesterase (AChE) assays in human blood show that the enzyme@nanoparticle construct decontaminates VX, preserving the biological function of AChE when exposed to an otherwise incapacitating dose. A porous silicon nanocage encapsulating phosphotriesterase variant L7ep‐3a is evaluated for nerve agent detoxification. By tuning the physicochemical properties of the enzyme–porous silicon nanoconstruct, enhanced catalytic activity, and operational stability are achieved compared to the free enzyme. Bolstered by its biocompatibility with human epidermal tissue, the nanobiocatalyst presents a strategy for nerve agent detoxification.