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Ene-reductases (ERs) are enzymes known for catalyzing the asymmetric hydrogenation of activated alkenes. Among these, old yellow enzyme (OYE) ERs have been the most extensively studied for biocatalytic applications due to their dependence on NADH or NADPH as electron donors. These flavin-containing enzymes are highly enantio- and stereoselective, making them attractive biocatalysts for industrial use. To discover novel thermostable OYE-type ERs, we explored genomes of thermophilic fungi. Five genes encoding ERs were selected and expressed in Escherichia coli, namely AtOYE (from Aspergillus thermomutatus), CtOYE (from Chaetomium thermophilum), LtOYE (from Lachancea thermotolerans), OpOYE (from Ogatae polymorpha), and TtOYE (from Thermothielavioides terrestris). Each enzyme was purified as a soluble FMN-containing protein, allowing detailed characterization. All ERs exhibited a preference for NADPH, with AtOYE showing the broadest substrate range. Moreover, all the enzymes showed activity toward maleimide and p-benzoquinone, with TtOYE presenting the highest catalytic efficiency. The optimal pH for enzyme activity was between 6 and 7 and the enzymes displayed notable solvent tolerance and thermostability, with CtOYE and OpOYE showing the highest stability (Tm > 60 °C). Additionally, all enzymes converted R-carvone into (R,R)-dihydrocarvone. In summary, this study contributes to expanding the toolbox of robust ERs. [ABSTRACT FROM AUTHOR]

Asymmetric hydrogenation of alkene moieties is important for the synthesis of chiral molecules, but achieving high stereoselectivity remains a challenge. Biocatalysis using ene-reductases (EReds) offers a viable solution. However, the need for NAD(P)H cofactors limits large-scale applications. Here, we explored an electrochemical alternative for recycling flavin-containing EReds using methyl viologen as a mediator. For this, we built a bio-electrocatalytic setup with an H-type glass reactor cell, proton exchange membrane, and carbon cloth electrode. Experimental results confirm the mediator's electrochemical reduction and enzymatic consumption. Optimization showed increased product concentration at longer reaction times with better reproducibility within 4-6 h. We tested two enzymes, Pentaerythritol Tetranitrate Reductase (PETNR) and the Thermostable Old Yellow Enzyme (TOYE), using different alkene substrates. TOYE showed higher productivity for the reduction of 2-cyclohexen-1-one (1.20 mM h -1 ), 2-methyl-2-cyclohexen-1-one (1.40 mM h -1 ) and 2-methyl-2-pentanal (0.40 mM h -1 ), with enantiomeric excesses ranging from 11 % to 99 %. PETNR outperformed TOYE in terms of enantioselectivity for the reduction of 2-methyl-2-pentanal (ee 59 % ± 7 % (S)). Notably, TOYE achieved promising results also in reducing ketoisophorone, a challenging substrate, with similar enantiomeric excess compared to published values using NADH., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C-C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R -selectivity of 83% ee . The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R -selectivity >99.9% ee . Michaelis-Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover k cat of 0.71 ± 0.03 s -1 and a K m of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Flavin mononucleotide (FMN)‐dependent ene‐reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon–carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene‐reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH‐preferring 12‐oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene‐reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β‐hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β‐hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β‐hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β‐hairpin flap favors NADH binding. [ABSTRACT FROM AUTHOR]

Flavors and fragrances (F&F) are interesting organic compounds in chemistry. These compounds are widely used in the food, cosmetic, and medical industries. Enzymatic synthesis exhibits several advantages over natural extraction and chemical preparation, including a high yield, stable quality, mildness, and environmental friendliness. To date, many oxidoreductases and hydrolases have been used to biosynthesize F&F. Ene-reductases (ERs) are a class of biocatalysts that can catalyze the asymmetric reduction of α,β -unsaturated compounds and offer superior specificity and selectivity; therefore, ERs have been increasingly considered an ideal alternative to their chemical counterparts. This review summarizes the research progress on the use of ERs in F&F synthesis over the past 20 years, including the achievements of various scholars, the differences and similarities among the findings, and the discussions of future research trends related to ERs. We hope this review can inspire researchers to promote the development of biotechnology in the F&F industry.

Repurposing enzymes to catalyze non-natural asymmetric transformations that are difficult to achieve using traditional chemical methods is of significant importance. Although radical C-O bond formation has emerged as a powerful approach for constructing oxygen-containing compounds, controlling the stereochemistry poses a great challenge. Here we present the development of a dual bio-/photo-catalytic system comprising an ene-reductase and an organic dye for achieving stereoselective lactonizations. By integrating directed evolution and photoinduced single electron oxidation, we repurposed engineered ene-reductases to steer non-natural radical C-O formations (one C-O bond for hydrolactonizations and lactonization-alkylations while two C-O bonds for lactonization-oxygenations). This dual catalysis gave a new approach to a diverse array of enantioenhanced 5- and 6-membered lactones with vicinal stereocenters, part of which bears a quaternary stereocenter (up to 99 % enantiomeric excess, up to 12.9 : 1 diastereomeric ratio). Detailed mechanistic studies, including computational simulations, uncovered the synergistic effect of the enzyme and the externally added organophotoredox catalyst Rh6G., (© 2024 Wiley-VCH GmbH.)

Enantioselective reduction of cyclobutenones to optically active cyclobutanones has been achieved by using whole‐cell overexpressing Seqenzym ene‐reductases (EREDs). By using these enzymes, trans‐cyclobutanones were isolated in good yields and with enantiomeric excesses up to 99%. [ABSTRACT FROM AUTHOR]

Enzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMN hq ) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.

Brazil’s territory encloses six terrestrial biomes (Amazon, Pantanal, Savannah, Atlantic Rainforest, and Caatinga-Savannah cactus) and a marine biome following the long coastline, yielding a wealth of native microbial species. Fungi found in different parts of Brazil are being studied for their enzymatic potential in biotransformation in organic synthesis processes. This review summarizes various studies on the bioreduction of activated C = C double bonds catalyzed by ene-reductases using fungi (filamentous and yeast) from the Brazilian environmental.

Repurposing enzymes to catalyze non‐natural asymmetric transformations that are difficult to achieve using traditional chemical methods is of significant importance. Although radical C−O bond formation has emerged as a powerful approach for constructing oxygen‐containing compounds, controlling the stereochemistry poses a great challenge. Here we present the development of a dual bio‐/photo‐catalytic system comprising an ene‐reductase and an organic dye for achieving stereoselective lactonizations. By integrating directed evolution and photoinduced single electron oxidation, we repurposed engineered ene‐reductases to steer non‐natural radical C−O formations (one C−O bond for hydrolactonizations and lactonization‐alkylations while two C−O bonds for lactonization‐oxygenations). This dual catalysis gave a new approach to a diverse array of enantioenhanced 5‐ and 6‐membered lactones with vicinal stereocenters, part of which bears a quaternary stereocenter (up to 99 % enantiomeric excess, up to 12.9 : 1 diastereomeric ratio). Detailed mechanistic studies, including computational simulations, uncovered the synergistic effect of the enzyme and the externally added organophotoredox catalyst Rh6G. [ABSTRACT FROM AUTHOR]

Thirteen Non-Conventional Yeasts (NCYs) have been investigated for their ability to reduce activated C=C bonds of chalcones to obtain the corresponding dihydrochalcones. A possible correlation between bioreducing capacity of the NCYs and the substrate structure was estimated. Generally, whole-cells of the NCYs were able to hydrogenate the C=C double bond occurring in (E)-1,3-diphenylprop-2-en-1-one, while worthy bioconversion yields were obtained when the substrate exhibited the presence of a deactivating electron-withdrawing Cl substituent on the B-ring. On the contrary, no conversion was generally found, with a few exceptions, in the presence of an activating electron-donating substituent OH. The bioreduction aptitude of the NCYs was apparently correlated to the logP value: Compounds characterized by a higher logP exhibited a superior aptitude to be reduced by the NCYs than compounds with a lower logP value.

Ene-reductases from the Old Yellow Enzyme (OYE) superfamily are a well-known and efficient biocatalytic alternative for the asymmetric reduction of C=C bonds. Considering the broad variety of substituents that can be tolerated, and the excellent stereoselectivities achieved, it is apparent why these enzymes are so appealing for preparative and industrial applications. Different classes of C=C bonds activated by at least one electron-withdrawing group have been shown to be accepted by these versatile biocatalysts in the last decades, affording a vast range of chiral intermediates employed in the synthesis of pharmaceuticals, agrochemicals, flavours, fragrances and fine chemicals. In order to access both enantiomers of reduced products, stereodivergent pairs of OYEs are desirable, but their natural occurrence is limited. The detailed knowledge of the stereochemical course of the reaction can uncover alternative strategies to orient the selectivity via mutagenesis, evolution, and substrate engineering. An overview of the ongoing studies on OYE-mediated bioreductions will be provided, with particular focus on stereochemical investigations by deuterium labelling., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)