ACS Catalysis

In Anabaena variabilis (Trichormus variabilis) phenylalanine ammonia-lyase (AvPAL), a conserved lid-like loop sits over the active site and has been studied both for its role in positioning a catalytic tyrosine and for its contribution to phenylalanine aminomutase (PAM) activity. While the active site architecture and substrate specificity of AvPAL have been extensively characterized, the dynamic behavior of this unstructured loop beyond its role in catalysis remains poorly understood. Here, we investigate the functional role of this loop by restricting its mobility through targeted interchain disulfide bond engineering. Three in-house approaches were designed to predict ideal cysteine residue pairs: (i) quantifying pair interaction energies via electrostatic and van der Waals forces, (ii) generating a contact map of residues within 5 [A] proximity, and (iii) implementing a machine-learning model trained on datasets from PDBCYS, SPX, and an internal database to rank cysteine pair likelihood within disulfide bond geometric constraints. Our machine-learning-guided strategy yielded a successful variant with complete oxidation efficiency in E. coli. Rigidification of this loop reveals that it also functions as a regulator of substrate specificity. Multi-scale molecular simulation analyses (molecular dynamics, metadynamics, quantum/molecular mechanics) reveal that this modification alters the active-site pocket by reducing the conformational dynamics of substrate binding. Our findings underscore the delicate balance between enzyme flexibility and catalytic efficiency, providing novel insights into the role of this understudied dynamic loop region in AvPAL.

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

Trimethylamine (TMA) is a major contributor to undesirable odours in protein hydrolysates derived from marine by-products, limiting their industrial use. Flavin-containing monooxygenases (FMOs) catalyse the conversion of TMA to the odourless trimethylamine N-oxide (TMAO); however, industrial applications demand enzymes that are both thermally stable and compatible with cost-effective cofactors. A thermostable variant of the Methylophaga aminisulfidivorans FMO (mFMO_20) can function at elevated temperatures but depends exclusively on the expensive and unstable cofactor NADPH. In this study, we investigated whether it is possible to simultaneously enhance thermostability and NADH compatibility using a multi-objective engineering strategy. We first targeted residues in the cofactor binding site of mFMO_20 to restore NADH activity, which had been completely lost despite the wild type enzyme being naturally active with both cofactors. Variants derived from the thermostable scaffold partially recovered NADH activity but showed reduced NADPH activity. Given the wild types inherent NADH compatibility, we next pursued a stability-improvement approach, introducing highly conserved stabilizing mutations. This preserved cofactor competence but produced only modest improvements in thermostability. Finally, by combining physical, evolutionary, and statistical metrics, we obtained variants that retained higher NADPH activity after heat treatment than any previously reported thermostable mutants, while a subset also retained measurable NADH activity before heat treatment. These findings show that combining complementary scoring strategies helps navigate the trade-off between stability and activity; while, robust NADH function under thermal stress remains elusive, with only one variant retaining detectable NADH activity after heat treatment, the results provide valuable insight into the underlying constraints linking stability and cofactor usage and highlights possible directions for engineering FMOs with both enhanced thermostability and cofactor compatibility. Author summaryIn this work, we aimed to improve an enzyme that could be useful in industrial applications but is limited by two common constraints: poor stability at high temperatures and dependence on an expensive cofactor. To make the enzyme more suitable for large-scale applications, we sought to engineer variants that are both more thermostable and compatible with a cheaper cofactor, NADH. For enzyme engineering, we used a strategy that balances several properties rather than prioritizing a single trait. We combined tools that capture evolutionary patterns, protein physics, and AI-based predictions to explore which mutations might provide the right combination of stability and function. Through this approach, we obtained variants with improved heat resistance and higher cofactor activity retention.

Glycoside hydrolases (GHs) play central roles in carbohydrate metabolism and are widely exploited for industrial and biomedical applications. However, they are often not optimal for applications due to their constrained function and strict stereochemical specificity, necessitating the discovery and optimization of distinct enzymes for each glycosidic configuration. Members of glycoside hydrolase family 1 (GH1) are archetypal retaining {beta}-glycosidases, while -specific activity is rare within this family. Here, I demonstrate that a retaining GH1 enzyme can be engineered to hydrolyze both {beta}- and -configured substrates without altering its canonical catalytic residues. Using a well-characterized {beta}-glycosidase and computational protein design strategies targeting second-shell residues surrounding the active site, a bifunctional {beta}-/-glycosidase containing 45 mutations was generated. The engineered variant acquired the ability to hydrolyze the -configured substrate 4-nitrophenyl--D-glucopyranoside while retaining activity toward the originals {beta}-substrates, with reduced catalytic efficiency and thermostability. Structural modeling and docking analyses reveal that the engineered enzyme preserves the original fold and accommodates substrates within the catalytic pocket in a similar manner to the wild type. These findings provide direct evidence that stereochemical constraint in retaining GH is more flexible than previously appreciated and can be modulated through targeted engineering.

The interdomain flexibility of GT-B fold glycosyltransferases regulates substrate binding and catalysis, yet the role of local structural variations in donor substrate specificity remains unclear. GumK, a GT70 enzyme from Xanthomonas campestris, exhibits local plasticity within its donor-binding pocket. We classify this plasticity into two conformational states, defined by the presence (closed state) or absence (open state) of a conserved hydrophobic interaction stabilizing the pocket. Using the AI-enhanced docking approach GNINA, we investigated the relationship between these states and substrate specificity by comparing UDP-glucuronate with various acidic and neutral substrate analogs. While docking scores showed limited discrimination among substrates, distance-based analysis between the sugar C6 atom and Lys307 revealed conformation-dependent trends. In the open state, negatively charged sugars preferentially interact with Lys307 via their carboxylate groups. Conversely, the closed state favors interactions with the pyrophosphate moiety. These results are consistent with physics-based simulations and suggest that donor specificity arises from the interplay between substrate chemistry and binding-site plasticity rather than from a single rigid binding mode. This study demonstrates how AI-driven docking, combined with an explicit representation of conformational states, can provide mechanistic insights into flexible enzymes and provide a rapid strategy for screening potential mutants.

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

Phosphoketolases can be used to convert non-phosphorylated sugars to the high energy compound acetyl phosphate and the versatile metabolic precursor acetyl-CoA. The performance of these pathways is limited by low catalytic activity of natural phosphoketolases towards these sugars. Here, we report the rational engineering of the phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) to enhance its activity and affinity towards glycoaldehyde (GA) and D-erythrulose (ERU) through re-organisation of the protein electric field to reproduce the role of terminal phosphate groups in cognate substrates. Guided by predicted induced side-chain pKa shifts, visualisation of electrostatic potential difference maps alongside molecular modelling and sequence variation analyses, we identified mutations that could promote in situ ring opening of the pre-dominant cyclic GA dimer form in solution. This approach to the electrostatic inverse design problem yielded the GA-specific double mutant H142N:E153D, exhibiting a ten-fold improved affinity and slightly enhanced catalytic efficiency (KM = 4.4 mM, kcat/ KM = 26.3 s-1 M-1) compared to the previously reported H142N variant (KM = 42.3 mM, kcat/ KM = 20.6 s-1 M-1). We additionally constructed a H256Y:H260Y:H548Y variant comprising long-range electrostatic mutations with a 3.8-fold increased catalytic efficiency (kcat/ KM = 49.6 s-1 M-1) on the acylic four-carbon ERU ketose compared to the wild-type enzyme. The engineered enzymes were evaluated in cell-free enzyme cascades for ATP regeneration via acetyl phosphate formation. The H142N variant enabled efficient ATP regeneration from GA and ethylene glycol, whereas the H142N:E153D mutant exhibited reduced stability under synthesis conditions. Furthermore, coupling of a highly GA-specific D-threose aldolase and a D-threose isomerase with the PKT triple mutant enabled rapid conversion of GA into C4 sugar intermediates and significantly improved ATP regeneration from GA.

Enzyme-directed evolution increasingly relies on computational tools to prioritize mutations, yet their practical value is difficult to assess because kinetic data are often aggregated across heterogeneous assay conditions, inflating apparent generalization. Here we introduce EnzyArena, a curated benchmark that groups kinetic parameters (kcat, Km, kcat/Km) into condition-matched experimental subsets to enable realistic evaluation. Using this resource, we benchmark 10 representative models from two arising strategy families--zero-shot fitness prediction and supervised kinetic-parameter prediction--across BRENDA- and SABIO-RK-derived subsets and 25 independent mutagenesis datasets. Kinetic-parameter predictors perform strongly on database-derived subsets but lose their advantage on independent datasets, whereas zero-shot predictors show more consistent generalization. A simple consensus of multiple zero-shot models further improves the precision of identifying beneficial mutants. We prospectively validated these findings in a wet-lab campaign (150 mutants) comparing random mutants, UniKP-prioritized mutants and ESM-1v-prioritized mutants (representing supervised kinetic-parameter prediction and zero-shot fitness prediction, respectively), where ESM-1v achieved the highest utility and UniKP underperformed the random baseline. Together, this study establishes realistic baselines for computational mutant prioritization and highlights consensus zero-shot strategies as a practical starting point for enzyme engineering.

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

Protein stabilization is a "Holy Grail" of biocatalysis, and stability design is an area of intense research interest. While it is increasingly feasible to effectively increase enzyme thermostability, optimization without compromising activity or selectivity remains a significant challenge. Here, we use full-atom protein sequence design with sidechain conditioning (FAMPNN) to engineer thermostable variants of the borneol dehydrogenase from Salvia rosmarinus (SrBDH1), an enzyme from a family where unselective enzymes dominate, and selectivity is determined by dynamical considerations. By combining FAMPNN design with residue conservation analysis and avoiding active site residues, we were able to computationally design SrBDH1 variants with up to 10 {degrees}C enhanced thermostability and strongly increased half-life time at elevated temperature, while retaining selectivity towards (+)-borneol. This design framework, integrating de novo and physics-based protein design tools, demonstrates that stability can be enhanced without disrupting functionally relevant dynamics, providing a route to engineer robust and selective biocatalysts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/719482v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@1a35073org.highwire.dtl.DTLVardef@f6c56dorg.highwire.dtl.DTLVardef@11b965forg.highwire.dtl.DTLVardef@2d6eef_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Genetic code expansion introduces new-to-nature chemical moieties into ribosomally synthesized proteins. In practice, the scope of functional groups that can be accessed using this method is often limited by noncanonical amino acid (ncAA) availability. Producing ncAAs directly in cells can circumvent poor ncAA uptake or commercial unavailability, but limited enzymes suitable for this application exist. In vitro evolution campaigns have been remarkably successful in yielding synthetically useful "ncAA synthases." However, these enzymes are optimized for preparative-scale synthesis and their activities often do not translate well to cellular biosynthesis. Thus, expanding strategies to engineer enzymes specifically for ncAA production within cells will benefit further implementation of genetic code expansion. Here, we use phage-assisted noncontinuous and continuous evolution to evolve enzymes for improved synthesis of non-canonical tyrosine derivatives in E. coli. Using simple serial passaging, we uncovered mutations that doubled the production of an expensive ncAA, 3-methoxytyrosine, by tyrosine phenol lyase, and furthermore evolved variants that enable 3-iodotyrosine biosynthesis, a transformation the parent enzyme is unable to catalyze. Additionally, we evolved a recently reported tyrosine synthase for improved production of 3-halogenated tyrosines, identifying variants that exhibit high activity even at low substrate concentrations owing to a [~]8-fold reduction in KM. Our results demonstrate that phage assisted evolution can be used to rapidly improve the activity of enzymes for ncAA production in cells.

Targeting the oxidative pentose phosphate pathway by inhibiting glucose-6-phosphate dehydrogenase (G6PD), is a promising anticancer strategy, yet clinically useful inhibitors remain unavailable. A key limitation is the lack of molecular insight into how allosteric and non-competitive inhibitors perturb enzymatic activity, limiting rational optimization. Here, we investigated the mechanism of G6PDi-1, a potent, reversible, non-competitive G6PD inhibitor, by combining biochemical assays with molecular dynamics simulations, Markov state model and MM/PBSA binding energy calculations. Experimentally, G6PDi-1 reduced active dimer formation in hepatoblastoma HepG2 cells and increased the inactive monomer fraction, linking inhibition to disrupted oligomerization. Computationally, we identified multiple sites on the enzyme surface, with preferential binding at dimer interface that sterically blocks oligomerization. Importantly, additional distal sites showed enhanced inter-residue correlations, suggesting secondary allosteric effects that shift G6PD toward monomeric states less competent for oligomerization. These findings provide a molecular basis for structure-based development of improved strategies for G6PD inhibition suited for cancer therapy.

Thermolysin, a bacterial zinc metalloprotease, has been previously been reported to exhibit a biphasic kinetic temperature dependence of kcat with a characteristic convex shape. This convex shaping is observed for almost all enzymes which display an Arrhenius break; fumarase is the exception with concave shaping. Here, thermolysin kinetics measured with the tripeptide substrate N-[3-(2-furyl)acryloyl]-Phe-Leu-Ala (FAFLA) resulted in a concave Arrhenius plot, characterized by a 30 kJ/mol increase in enthalpy and entropy of activation, in contrast to the typical 30 kJ/mol decrease. Although the shape of the Arrhenius break differs, ionic strength and macromolecular crowding both attenuate the energetic magnitude of the break point, consistent with prior work. It was hypothesized that a different step of the catalytic cycle of thermolysin was represented by kcat with FAFLA to give rise to this new behavior. A 91% dependence of kcat on viscosity and modest solvent isotope effects, both distinct from previously-characterized substrates, indicated that a physical step was responsible for the observed Arrhenius concavity. Hinge bending conformational changes of thermolysin, monitored using the phosphoramidon inhibitor (a FAFLA mimic), exhibited a fully linear temperature dependence, excluding these large-scale motions as the origin of concavity. It was therefore proposed that release of the N-[3-(2-furyl)acryloyl]-Phe product is likely rate limiting since release was proposed to involve a two-step pathway to free the product coordinated to the catalytic Zn2+ of thermolysin. These findings provide a mechanistic framework for seldom-seen concave break point behavior and insights into the contribution of dynamics of physical processes to catalysis. IMPORTANCE AND IMPACTEnzymes which display Arrhenius break behavior provide insight into how dynamics impact catalysis. Almost every enzyme thus far displays convex biphasic shape, with concave shaping often not acknowledged. Thermolysin, which previously only showed convex shaping, displayed concave behavior with a tripeptide substrate. By linking this unusual kinetic behavior to a physical, not chemical, process, this work highlights the possible origin of a rare phenomenon which can expand understanding of protein dynamics and biphasic Arrhenius behavior.

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of Nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

Thiol dioxygenases (TDOs) catalyze the incorporation of molecular oxygen into thiol metabolites and N-terminal cysteine residues of regulatory proteins, thereby playing critical roles in sulfur metabolism and oxygen sensing. Despite extensive study over the past two decades, the molecular basis for substrate recognition and the catalytic mechanism of TDOs remains controversial, owing to the scarcity of substrate-bound structures and direct evidence for catalytic intermediates. Herein, we present a comprehensive study of mercaptosuccinate dioxygenase (MSDO), a TDO originally identified in Variovorax paradoxus B4, using a combination of structural, biochemical, spectroscopic, and computational approaches. MSDO oxidizes both (S)- and (R)-mercaptosuccinate (MS) with similar Km values but exhibits approximately 2.5-fold higher turnover for the (S)-enantiomer. Crystal structures of MSDO reveal that both (S)- and (R)-MS coordinate the iron in a bidentate mode via their thiolate and proximal carboxylate groups, with the distal carboxylate adopting distinct orientations. Two active-site Arg residues recognize the substrate carboxylate groups and thereby stabilize a flexible C-terminal loop, underpinning a catalytic site gating mechanism in MSDO. EPR spectroscopy corroborates bidentate coordination, showing conversion of a high-spin {FeNO}7 complex to a low-spin species upon substrate binding. Time-resolved in crystallo reactions capture two key iron-bound intermediates, namely an unprecedented monooxygenated sulfenate and a dioxygenated sulfinate product. These structural snapshots are supported by DFT calculations that point to a stepwise oxygen atom transfer pathway. Computational analysis further accounts for the kinetic differences between the substrate enantiomers, as rationalized by structural comparisons, active-site geometry, and second coordination sphere interactions. Together, these results elucidate fundamental principles of TDO catalysis and advance our understanding of nonheme iron-dependent oxygen activation.

The radical S-adenosylmethionine (SAM) superfamily comprises more than 800,000 enzymes that use [Fe4S4] clusters to initiate radical chemistry that mediates an exceptionally broad range of chemical transformations. Within this superfamily, cobalamin (Cbl)-dependent radical SAM enzymes constitute a major subclass predominantly associated with methylation reactions. However, several notable members catalyze non-methylase reactions, for which the mechanistic role of Cbl is poorly understood. Bacteriochlorophyll biosynthesis enzyme BchE is a Cbl-dependent radical SAM enzyme that catalyzes a six-electron oxidation of Mg-protoporphyrin IX monomethylester (MPE) to protochlorophyllide (PChlide), installing a ketone and forming the fifth ring of bacteriochlorophyll under anaerobic conditions. Although prior in vivo and in vitro studies have demonstrated a requirement for Cbl, SAM, and a low-potential reductant, detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme. Here, we report the successful isolation and spectroscopic characterization of BchE, enabling the first in vitro reconstitution of its enzymatic activity. Using both chemical and biological reducing systems, we observe the formation of PChlide along with proposed reaction intermediates and several off-pathway products. These results provide new insight into the oxidative chemistry mediated by Cbl in non-methylase radical SAM enzymes and establish BchE as a tractable model for elucidating how cobalamin is deployed in this understudied subclass.

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H2, making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O2 sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani, has exhibited exceptional O2-stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O2-induced degradation by formation of the Hinact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe]H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the Hinact and Htrans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydAPDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fed center of [2Fe]H, thereby reducing Hinact accumulation. Moreover, as-isolated ToHydAPDT predominantly exhibits the Hhyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe]H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.

O2-tolerance is a desirable property for [FeFe] hydrogenases, which are highly efficient H2-producing catalysts. While most such enzymes are highly sensitive to aerobic environments, a small number of explored representatives exhibit exceptional stability and even H2-producing activity under oxygenic conditions. However, the genetic signatures of the O2-tolerance in this class of enzymes remain largely unknown. To address this knowledge gap, we explored a close homologue of a well-characterized O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) - a hydrogenase from Terrisporobacter glycolicus (TgHydA1). Our investigation indeed confirms that TgHydA1 can transition to the O2-stable Hinact state, a hallmark of O2 tolerance. The surprising outcome is that despite the high amino acid similarity, TgHydA1 shows a substantially higher propensity to remain in the Hinact state than CbHydA1. Using protein film electrochemical experiments, we demonstrate that the root of this behavior lies in roughly tenfold slower reactivation rates than those of CbHydA1 at any applied potential. This degree and direction of variation in reactivation kinetics have not been observed before for any other O2-tolerant [FeFe] hydrogenases or their variants to date, uncovering a yet-to-be-explored facet of reactivity alteration available to these enzymes. Overall, the results presented here highlight the importance of a holistic analysis of [FeFe] hydrogenase sequences in the context of their interaction with O2 that encompasses the protein environment and properties of the auxiliary metallocofactors.

Directed evolution has long been constrained by complex screening hardware and labor-intensive workflows. Here, we report the first genuine test-tube screening platform that uses His6-tagged peptide-functionalized magnetic beads and Fe3+-decorated E. coli cells to establish a phenotype-genotype linkage, thereby decoupling ultrahigh-throughput screening from specialized instrumentation and democratizing directed evolution. The platform demonstrated a screening throughput of > 108 events s-1 and an enrichment factor of up to 63-fold. Using galactose oxidase as a model, we identified variants with up to a 26-fold increase in catalytic efficiency. Extensions to D-amino acid oxidase and alcohol oxidase yielded variants with up to 5383-fold and 25-fold improvements over their respective wildtypes after a single round of screening. These results highlight the platforms capacity to rapidly engineer H2O2-generating oxidases and to advance AI-driven enzyme design through rapid data generation.