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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]

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]

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]

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]

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]

Brivaracetam is an effective third-generation antiepileptic drug, and its two chiral centers are a major challenge for designing an inexpensive large-scale synthesis process. Enzymatic asymmetric reduction offers an alternative biotransformation method for reducing 2-(5H)-furanone-4-propyl (3a) to (R)-4-propyldihydrofuran-2(3 H)-one (3b), which is a key intermediate in synthesizing brivaracetam. However, no enzymes have been reported to catalyze this reaction with both high activity and stereoselectivity. In this study, fourteen ene-reductases were compared, and the old yellow enzyme OYE2 was identified as having strict stereoselectivity but unsatisfactory activity. Through evaluations with structurally similar substrates and alanine-screening methods, two key residues were identified, T37 and Y82, playing critical roles in catalytic activity. The hydrogenation activity of mutants Y82W and T37C increased by 2.97 and 7.13 times, respectively. Molecular dynamics simulation indicates that mutant Y82W might shorten the distance between the electron acceptor and donor, while T37A might decrease the redox potential of FMN and then accelerate hydrogen transfer. These results indicate that tuning the redox potential properties of FMN is an important consideration for engineering FMN-dependent oxidoreductases. During the gram-scale synthesis of 3b , T37C exhibited excellent stereoselectivity (>99 % ee) and conversion (>99 %), laying a foundation for a simple one-step route in producing the chiral precursor brivaracetam. [Display omitted] • A brivaracetam precursor was synthesized by old yellow enzyme OYE2. • Two key points, Thr37 and Tyr82, were identified with enhanced reduction activities. • Mutation in Tyr82 site could shorten the distance between electron donor and acceptor. • Mutation in Thr37 site could decrease the redox potential of flavin mononucleotide. [ABSTRACT FROM AUTHOR]

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis. [ABSTRACT FROM AUTHOR]

Most ene‐reductases belong to the Old Yellow Enzyme (OYE) family of flavin‐dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron‐withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X‐ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual‐specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a β‐hairpin motif in an active site loop of both these enzymes were studied using stopped‐flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the β‐hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15‐fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55‐fold) increase in efficiency in the reductive half‐reaction, when compared to the essentially unreactive wild‐type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs. [ABSTRACT FROM AUTHOR]

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom‐economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene‐reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min−1 and total turnover numbers up to 20 200 were recorded during flavin recycling. [ABSTRACT FROM AUTHOR]

A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H2 as a simple, completely atom‐economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH2 or FADH2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme "ene‐reductases" to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min−1 and total turnover numbers up to 20 200 were recorded during flavin recycling. [ABSTRACT FROM AUTHOR]

Flavin‐dependent 'ene'‐reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be "dynamically stable", suggesting it is sufficiently long‐lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models. [ABSTRACT FROM AUTHOR]

Flavin‐dependent 'ene'‐reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be "dynamically stable", suggesting it is sufficiently long‐lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models. [ABSTRACT FROM AUTHOR]

Flavin‐dependent ene‐reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride‐transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non‐natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single‐electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis. [ABSTRACT FROM AUTHOR]

Flavin‐dependent ene‐reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride‐transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non‐natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single‐electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis. [ABSTRACT FROM AUTHOR]

Chiral carveol and dihydrocarveol are important additives in the flavor industry and building blocks in the synthesis of natural products. Despite the remarkable progress in asymmetric catalysis, convenient access to all possible stereoisomers of carveol and dihydrocarveol remains a challenge. Here, we present the stereodivergent synthesis of carveol and dihydrocarveol through ketoreductases/ene‐reductases catalyzed asymmetric reduction. By directly asymmetric reduction of (R)‐ and (S)‐carvone using ketoreductases, which have Prelog or anti‐Prelog stereopreference, all four possible stereoisomers of carveol with medium to high diastereomeric excesses (up to >99 %) were first observed. Then four stereoisomers of dihydrocarvone were prepared through ene‐reductases catalyzed diastereoselective synthesis. Asymmetric reduction of obtained dihydrocarvone isomers by ketoreductases further provide access to all eight stereoisomeric dihydrocarveol with up to 95 % de values. In addition, the absolute configurations of dihydrocarveol stereoisomers were determined by using modified Mosher's method. Tasty, tasty: By directly asymmetric reduction of (R)‐ and (S)‐carvone using ketoreductases, all four possible stereoisomers of carveol with medium to high diastereomeric excesses (up to >99 %) were first observed. Then previously studied and newly engineered ene‐reductases were applied to synthesis four stereoisomers of dihydrocarvone. Asymmetric reduction of obtained dihydrocarvone isomers by ketoreductases further provide access to all eight stereoisomeric dihydrocarveol. [ABSTRACT FROM AUTHOR]

Abstract: Ene reductases from the Old Yellow Enzyme (OYE) family reduce the C=C double bond in α,β‐unsaturated compounds bearing an electron‐withdrawing group, for example, a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we show that members of this enzyme family can also catalyze the formation of C−C bonds. α,β‐Unsaturated aldehydes and ketones containing an additional electrophilic group undergo reductive cyclization. Mechanistically, the two‐electron‐reduced enzyme cofactor FMN delivers a hydride to generate an enolate intermediate, which reacts with the internal electrophile. Single‐site replacement of a crucial Tyr residue with a non‐protic Phe or Trp favored the cyclization over the natural reduction reaction. The new transformation enabled the enantioselective synthesis of chiral cyclopropanes in up to >99 % ee. [ABSTRACT FROM AUTHOR]

A series of functionalized α-chiral phosphonates was obtained by enzymatic reduction of the corresponding β-activated vinylphosphonate derivatives employing several ene-reductases of the Old Yellow Enzyme family as biocatalysts. The insufficient activation of the phosphonate group alone was compensated by introduction of an electron-withdrawing group at the β-position, which resulted in high levels of conversion (up to >99%). All active enzymes consistently furnished ( R)-configurated products with exquisite stereoselectivity, thereby providing a stereo-complementary approach to current asymmetric hydrogenation protocols on similar compounds. Preparative-scale syntheses performed in aqueous buffer using formate/formate dehydrogenase for recycling of the nicotinamide cofactor granted access to β-keto-, cyano- and ester phosphonates from the ( E)-isomers of α,β-unsaturated phosphonates in up to 72% isolated yield and >99% ee. [ABSTRACT FROM AUTHOR]

In contrast to the widely studied asymmetric bioreduction of α,β-unsaturated carboxylic acid esters catalyzed by ene-reductases, the reaction applied to lactones remains unexplored. A broad set of ene-reductases was found to reduce various α-, β- and γ-substituted α,β-unsaturated butyrolactones to yield the corresponding saturated non-racemic lactones. Substitution patterns greatly influenced activities and stereoselectivities and lactone products were obtained in moderate to excellent yields; importantly, enzyme-based stereocontrol allowed access to both enantiomers in up to >99% ee. Chiral recognition of a distant γ-center led to kinetic resolution with remarkable enantioselectivities (E values up to 49). An unprecedented case of dynamic kinetic resolution was observed with 3-methyl-5-phenylfuran-2(5 H)-one, whereby spontaneous racemization of the substrate furnished the product in up to 73% conversion and >99% ee and 96% de. [ABSTRACT FROM AUTHOR]

Flavin has long been known to function as a single electron reductant in biological settings, but this reactivity has rarely been observed with flavoproteins used in organic synthesis. Here we describe the discovery of an enantioselective radical dehalogenation pathway for α-bromoesters using flavin-dependent ‘ene’-reductases. Mechanistic experiments support the role of flavin hydroquinone as a single electron reductant, flavin semiquinone as the hydrogen atom source, and the enzyme as the source of chirality. [ABSTRACT FROM AUTHOR]

The asymmetric bioreduction of α,β-unsaturated γ-keto esters using ene-reductases from the Old Yellow Enzyme family proceeds with excellent stereoselectivity and high conversion levels, covering a broad range of acyclic and cyclic derivatives. Various strategies were employed to provide access to both enantiomers, which are versatile precursors of bioactive molecules. The regioselectivity of hydride addition on di-activated alkenes was elucidated by isotopic labeling experiments and showed strong preference for the keto moiety as activating/binding group as opposed to the ester. Finally, chemoenzymatic synthesis of (R)-2-(2-oxocyclohexyl)acetic acid was achieved in high ee on a preparative scale combining enzymatic reduction followed by ester hydrogenolysis. [ABSTRACT FROM AUTHOR]

Ene-reductases are flavoproteins able to catalyse the reduction of carbon-carbon double bonds with many potential applications in biocatalysis. The fungus Mucor circinelloides MUT44 has high ene-reductase activity when grown in the presence of substrates carrying different electron-withdrawing groups. Genome sequencing revealed the presence of ten putative genes coding for ene-reductases that can be potentially exploited for biocatalytic purposes. To this end, the availability of a method able to predict which isoform binds and turns over a specific substrate would help to choose the best catalyst for the desired bioconversion. Here, homology models of the ten putative enzymes are first generated, validated and show that the proteins share the typical TIM barrel fold with a conserved β-hairpin cap on one side of the barrel and a non-conserved subdomain capping the other side, where the FMN cofactor is accommodated. The active site of the ten enzymes is different in terms of both volume and charge distribution whereas the residues responsible for substrate recognition and catalysis are generally conserved. Docking of cyclohexenone into the active site of the ten enzymes shows a binding almost superimposable to that found in pentaerythritol tetranitrate reductase in complex with this substrate (PDB ID 1GVQ ) in isoforms 1, 2, 6 and 10. The data demonstrate that in silico predictions can be used for new putative fungal ene-reductases to predict the best substrate-enzyme matching for the selection of the most suitable catalyst for the desired biotransformation. [ABSTRACT FROM AUTHOR]

One-pot sequential and cascade processes performed by employing ene-reductases (ERs) together with ω-transaminases (ω-TAs) for the obtainment of diastereomerically enriched ( R)- and ( S)-amine derivatives containing an additional stereocenter were investigated. By using either α- or β-substituted unsaturated ketones as substrates and by coupling purified ERs belonging to the Old Yellow Enzyme (OYE) family with a panel of commercially available ω-TAs, the desired products were obtained in up to >99 % conversion and >99 % de. The sequential reactions were performed in a one-pot fashion with no need to adapt the reaction conditions to the reductive amination step or to purify the reaction intermediate. Moreover, high chemoselectivity of the tested ω-TAs for the saturated ketones was shown in the cascade reactions. [ABSTRACT FROM AUTHOR]

In this study an evaluation of the synthetic potential of a broad range of recombinant ene reductases was performed. In detail, a library of 23 ene reductases was used to screen the CC reduction of 21 activated alkenes from different compound classes as substrates. The chosen set of substrates comprises nitroalkenes with an aryl substituent at the β-position and a methyl substituent at the α- or β-position, α,β-unsaturated carboxylic acids and their esters with and without substituents at the β-position, a range of cyclic α,β-unsaturated ketones with different ring sizes and substitution patterns and one α,β-unsaturated boronic acid. After we obtained insight into the substrate scope, several biotransformations were prioritised and further investigated in a screening of 41 reaction parameters (which included chaotropic and kosmotropic salts, polyols, buffer solutions, amino acids and organic solvents) towards their impact on the activity and enantioselectivity of the applied ene reductases. Under the optimised conditions, selected reduction processes were performed on an increased lab scale (up to 30 mL) with up to 10 % substrate concentration, which led in general to both high conversion and (if chiral products were formed) enantioselectivity. [ABSTRACT FROM AUTHOR]

The enantioselective reduction of α-methylene nitrile derivatives catalysed by ene-reductases affords the corresponding ( R)-2-arylpropanenitriles with high conversion values. The reaction is investigated either in aqueous medium (with an organic cosolvent or by loading the substrate onto hydrophobic resins) or in a biphasic ionic liquid-water system. The use of ionic liquids, herein with isolated ene-reductases, is found to improve the work-up and the substrate recovery method. The synthetic manipulation of the final chiral nitrile derivatives indicates how this biocatalysed method can be exploited for the preparation of a wide range of chiral compounds. [ABSTRACT FROM AUTHOR]

To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two new ene-reductases cloned and characterized. [•] Reduction shown for a variety of enones, dimethylesters, aldehyde and maleimdes. [•] Asymmetric reduction of the isomers of carvone, ketoisophorone and 2-methylcyclohexenone. [•] NAD(P)H-free light-driven biocatalysis. [Copyright &y& Elsevier]

By integrating enzymatic catalysis with photocatalysis, photoenzymatic catalysis emerges as a powerful strategy to enhance enzyme catalytic capabilities and provide superior stereocontrol in reactions involving reactive intermediates. Repurposing naturally occurring enzymes using visible light is among the most active directions of photoenzymatic catalysis. This Minireview focuses on a cutting‐edge strategy in this direction, namely single‐electron‐oxidation‐triggered non‐natural biotransformations catalyzed by photoexcited enzymes. These straightforward transformations feature a unique radical mechanism initiated by single‐electron oxidation, achieving redox‐neutral non‐natural C−C, C−O, and C−S bond formation, and expanding the chemical toolbox of enzymes. By highlighting recent advances in this field and emphasizing their catalytic mechanisms and synthetic potential, innovative approaches for photobiomanufacturing are anticipated. [ABSTRACT FROM AUTHOR]

The asymmetric bioreduction of a library of β-cyanoacrylate esters using ene-reductases was studied with the aim to provide a biocatalytic route to precursors for GABA analogues, such as pregabalin. The stereochemical outcome could be controlled by substrate-engineering through size-variation of the ester moiety and by employing stereochemically pure (E)- or (Z)-isomers, which allowed to access both enantiomers of each product in up to quantitative conversion in enantiomerically pure form. In addition, stereoselectivities and conversions could be improved by mutant variants of OPR1, and the utility of the system was demonstrated by preparative-scale applications. [ABSTRACT FROM AUTHOR]

• In-detailed identification of four ene reductases. • The biochemical and kinetic properties of the mined ene reductases were characterized to understand their catalytic power. • The four mined ene reductases were applied to synthesize the dihydrocarvones and profen derivatives. • Molecular simulation was performed to understand the origin of enzymes' activity and stereoselectivity. The ene reductases (ERs) from the old yellow enzymes (OYEs) family have the ability to reduce activated alkenes to generate up to two stereocenters, therefore they have been received extensive attention as powerful biocatalysts. In this study, through gene mining, four ERs were identified from the genomes of Ensifer adhaerens , Pseudomonas fluorescens , and Pseudomonas veronil. The biocatalytic properties of these four ERs were identified, and their applications in the synthesis process of dihydrocarvone and profen derivatives were further evaluated. Among them, three ERs (EaER2, PvER1, and PvER2) belonging to the classic OYEs showed the best catalytic activity at 30 °C and pH 7.0 (100 mM potassium phosphate buffer) and the PfER2, which belongs to the thermophilic-like OYEs exhibited the best catalytic at 40 °C and pH 7.0 (100 mM potassium phosphate buffer). When exploring the influence of organic solvents on the catalytic efficiency, it was found that the four ERs were more sensitive to toluene and had tolerance to several other selected organic solvents. In addition, EaER2, PfER2, PvER1 and PvER2 showed excellent catalytic activity toward carvone, and the stereoselectivity of PvER2 toward carvone could reach up to 88.7 % de. EaER2 and PfER2 can catalyze the synthesis of a variety of profen derivatives with a stereoselectivity over 99 % ee. Moreover, through homology modeling and molecular docking, we preliminarily explained the mechanism of catalytic activity and stereoselectivity of the four ERs, which provided a solid base on the rational design of their stereo-preference in the future. The discovery of EaER2, PfER2, PvER1, and PvER2 provides four new enzyme sources for the study of the OYEs family and enriches the biocatalytic toolbox of ERs. Our exploration of the enzymatic properties of these four ERs will provide the sufficient data basis for future research and industrialization progress. [ABSTRACT FROM AUTHOR]

Four NAD(P)H-dependent non-flavin ene reductases have been investigated for their ability to reduce activated C=C bonds in an asymmetric fashion by using 20 structurally diverse substrates. In comparison with flavin-dependent Old Yellow Enzyme homologues, a higher degree of electronic activation was required, because the best activities were obtained with enals and nitroalkenes rather than enones and carboxylic esters. Although FaEO from Fragaria x ananassa (strawberry) and its homologue SlEO from Solanum lycopersicum (tomato) exhibited a narrow substrate spectrum, progesterone 5β-reductase (At5β-StR) from Arabidopsis thaliana (thale cress) and leukotriene B4 12-hydroxydehydrogenase (LTB4DH/PGR) from Rattus norvegicus (rat) appear to be promising candidates, in particular for the asymmetric bioreduction of open-chain enals, nitroalkenes and α,β-unsaturated γ-butyrolactones. Competing nitro reduction and non-enzymatic Weitz-Scheffer epoxidation were largely suppressed. [ABSTRACT FROM AUTHOR]

The baker's yeast (BY)-mediated reduction of a set of α-substituted derivatives of cinnamaldehyde to give the corresponding saturated alcohols was investigated. Our study demonstrates that the ene reductases of BY (OYE 2 or OYE 3) preferentially reduce the CC double bond of stereoisomers with the phenyl group trans to the carbonyl group. Moreover, the alcohol dehydrogenases (ADHs) chemoselectively reduce (oxidise) the carbonyl group (hydroxyl group) of saturated aldehydes (alcohols) if the side chain does not contain a heteroatom such as oxygen or sulphur and is longer than an ethyl group. Using isolated OYEs instead of BY, the saturated aldehydes partially lost their optical purity during the biotransformation, even with the in situ substrate feeding product removal (SFPR) technology. This limitation can be overcome by the combination of an isolated ene reductase (OYE 2 or OYE 3) together with an ADH (horse liver alcohol dehydrogenase) in a cascade system which allowed both yields and enantioselectivities to improve. [ABSTRACT FROM AUTHOR]

Hydride source: Commercially viable biocatalytic reductions must overcome the cost barrier of NAD(P)H requirements. Developments in overcoming the “hydride problem” are discussed in this Highlight. [ABSTRACT FROM AUTHOR]

(2R,5R)-dihydrocarvone is an industrially applied building block that can be synthesized by site-selective and stereo-selective C=C bond bio-reduction of (R)-carvone. Escherichia coli (E.coli) cells overexpressing an ene reductase from Nostoc sp. PCC7120 (NostocER1) in combination with a cosubstrate regeneration system proved to be very effective biocatalysts for this reaction. However, the industrial applicability of biocatalysts is strongly linked to the catalysts' activity. Since the cell-internal NADH concentrations are around 20-fold higher than the NADPH concentrations, we produced E.coli cells where the NADPH-preferring NostocER1 was exchanged with three different NADH-accepting NostocER1 mutants. These E. coli whole-cell biocatalysts were used in batch operated stirred-tank reactors on a 0.7 l-scale for the reduction of 300 mM (R)-carvone. 287 mM (2R,5R)-dihydrocarvone were formed within 5 h with a diasteromeric excess of 95.4% and a yield of 95.6%. Thus, the whole-cell biocatalysts were strongly improved by using NADH-accepting enzymes, resulting in an up to 2.1-fold increased initial product formation rate leading to a 1.8-fold increased space-time yield when compared to literature. [ABSTRACT FROM AUTHOR]

Asymmetric bioreduction of an ( E)-β-cyano-2,4-dienoic acid derivative by ene-reductases allowed a shortened access to a precursor of pregabalin [( S)-3-(aminomethyl)-5-methylhexanoic acid] possessing the desired configuration in up to 94% conversion and >99% ee. Deuterium labelling studies showed that the nitrile moiety was the preferred activating/anchor group in the active site of the enzyme over the carboxylic acid or the corresponding methyl ester. [ABSTRACT FROM AUTHOR]

Ene reductases (ERs) are attractive biocatalysts in terms of their high enantioselectivity and expanded substrate scope. Recent works have proved that synthetic nicotinamide coenzyme biomimetics (NCBs) can be used as easily accessible alternatives to natural cofactors in ER‐catalyzed reactions. However, the structure‐reactivity relationships between NCBs and ERs and influence factors are still poorly understood. In this study, a series of C‐5 methyl modified NCBs were synthesized and tested in the PETNR‐catalyzed asymmetric reductions. The physicochemical properties of these NCBs including electrochemical properties, stability, and kinetic behavior were studied in detail. The results showed that hydrophobic interaction caused by the introduced methyl group contributed to the stabilization of binding conformation in enzyme active site, resulting in comparable catalytic activity with that of NADPH. Molecular dynamics and steered molecular dynamics simulations were further performed to explain the binding mechanism between PETNR and NCBs, which revealed that stable catalytic conformation, appropriate donor‐acceptor distance and angle, as well as free dissociation energy are important factors affecting the activity of NCBs. [ABSTRACT FROM AUTHOR]

The front cover image, provided by Piotr Kiełbasiński, Mélanie Hall and collaborators, illustrates the stereoselective bioreduction of activated vinylphosphonates catalyzed by Old Yellow Enzyme homologues, which can be easily produced through recombinant expression in E. coli cells. This stereocomplementary method to current asymmetric hydrogenation protocols provides access to α‐chiral phosphonates of high enantiomeric purity. (Picture from E. coli bacteria – NIAID; Under Creative Common license BY‐SA). Details can be found in the full paper on pages 4190–4196 (I. Janicki, P. Kiełbasiński, N. G. Turrini, K. Faber, M. Hall, Adv. Synth. Catal. 2017, 359, 4190–4196; DOI: 10.1002/adsc.201700716). [ABSTRACT FROM AUTHOR]

The article presents chemical equations related to the article "Enzymatic Synthesis of Optically Active Lactones via Asymmetric Bioreduction Using Ene-Reductases from the Old Yellow Enzyme Family" by N.G. Turrini and others, published in the periodical "Advanced Synthesis and Catalysis" in 2015.

The direct synthesis of alkenes from alkynes usually requires the use of transition‐metal catalysts. Unfortunately, efficient biocatalytic alternatives for this transformation have yet to be discovered. Herein, the selective bioreduction of electron‐deficient alkynes to alkenes catalysed by ene‐reductases (EREDs) is described. Alkynes bearing ketone, aldehyde, ester, and nitrile moieties have been effectively reduced with excellent conversions and stereoselectivities, observing clear trends for the E/Z ratios depending on the nature of the electron‐withdrawing group. In the case of cyanoalkynes, (Z)‐alkenes were obtained as the major product, and the reaction scope was expanded to a wide variety of aromatic substrates (up to >99 % conversion, and Z/E stereoselectivities of up to >99/1). Other alkynes containing aldehyde, ketone, or ester functionalities also proved to be excellent substrates, and interestingly gave the corresponding (E)‐alkenes. Preparative biotransformations were performed on a 0.4 mmol scale, producing the desired (Z)‐cyanoalkenes with good to excellent isolated yields (63–97 %). This novel reactivity has been rationalised through molecular docking by predicting the binding poses of key molecules in the ERED‐pu‐0006 active site. [ABSTRACT FROM AUTHOR]

The direct synthesis of alkenes from alkynes usually requires the use of transition‐metal catalysts. Unfortunately, efficient biocatalytic alternatives for this transformation have yet to be discovered. Herein, the selective bioreduction of electron‐deficient alkynes to alkenes catalysed by ene‐reductases (EREDs) is described. Alkynes bearing ketone, aldehyde, ester, and nitrile moieties have been effectively reduced with excellent conversions and stereoselectivities, observing clear trends for the E/Z ratios depending on the nature of the electron‐withdrawing group. In the case of cyanoalkynes, (Z)‐alkenes were obtained as the major product, and the reaction scope was expanded to a wide variety of aromatic substrates (up to >99 % conversion, and Z/E stereoselectivities of up to >99/1). Other alkynes containing aldehyde, ketone, or ester functionalities also proved to be excellent substrates, and interestingly gave the corresponding (E)‐alkenes. Preparative biotransformations were performed on a 0.4 mmol scale, producing the desired (Z)‐cyanoalkenes with good to excellent isolated yields (63–97 %). This novel reactivity has been rationalised through molecular docking by predicting the binding poses of key molecules in the ERED‐pu‐0006 active site. [ABSTRACT FROM AUTHOR]

Synthetic nicotinamide biomimetics (NCBs) have emerged as alternatives to the use of natural cofactors. The relatively low cost and ease of manufacture of NCBs may enable the scaling of biocatalytic reactions to produce bulk chemicals (e.g., biofuels and plastics). NCBs are also recognized by only a subset of NAD(P)/NAD(P)H-dependent enzymes, which potentially allows access to orthogonal redox cascades that can be run simultaneously within a single reactor. In the work presented here, a series of NCBs was prepared and tested for activity with alcohol dehydrogenases and ene-reductases. While the NCBs did not support enzymatic activity with the alcohol dehydrogenases, the observed rate of the ene-reductases with NCBs was greater than when incubated with the natural cofactor (consistent with previous observations). We obtained the structures of an ene-reductase and an alcohol dehydrogenase with an NCB bound in their active sites. While the NCB bound to the ene-reductases in a productive position and orientation for hydride transfer to the isoalloxazine ring of the flavin cofactor, the NCB failed to adopt a catalytically competent binding mode in the alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

The article presents chemical equations related to the article "NAD(P)H-Independent Asymmetric C=C Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co-Substrates as the Hydrogen Donor" by C.K. Winkler and others, published in the "Chemistry-A European Journal" in 2014.

γ‐Amino esters and γ‐lactams containing α,γ‐stereogenic centers are widely used as chiral intermediates in various bioactive compounds, while their efficient synthesis remains a challenge. Herein, an enzymatic cascade reaction involving an ene reductase (ERED) and an imine reductase (IRED) for accessing to α,γ‐stereospecific γ‐amino esters and γ‐lactams from α,β‐unsaturated γ‐ketoesters has been developed. 21 EREDs and 13 IREDs have been identified with high activity and stereoselectivity toward various α,β‐unsaturated γ‐ketoesters and the corresponding chiral γ‐ketoesters, respectively. By employing suitable ERED and IRED, (1S,3S), (1S,3R), (1R,3S), and (1R,3R) stereoisomers of methyl 3‐(cyclopropyl amino) cyclohexane‐1‐carboxylate were obtained as a single stereoisomer in 63–72% yield, and (3R,5R)‐ and (3R,5S)‐1‐cyclopropyl‐3,5‐dimethylpyrrolidin‐2‐one and 1‐cyclopropyl‐3,5‐dipropylpyrrolidin‐2‐one were also prepared as a single stereoisomer in 60–65% yields. This study establishes an environmentally friendly enzymatic route for the synthesis of γ‐amino esters and γ‐lactams containing α,γ‐stereogenic centers. [ABSTRACT FROM AUTHOR]

Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non‐natural reactivity in enzymes to solve long‐standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp3−Csp3 bonds with high chemo‐ and enantioselectivity. [ABSTRACT FROM AUTHOR]

Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non‐natural reactivity in enzymes to solve long‐standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp3−Csp3 bonds with high chemo‐ and enantioselectivity. [ABSTRACT FROM AUTHOR]

The development of more efficient and environmentally friendly analytical methods represents a current focus for the fine chemical industry. In particular, microscale methodologies that are free of solvents/reagents. The headspace-GC/MS (HS-GC) methodology was employed in this study as a tool for monitoring a biocatalysed reaction of (4S)-(+)-carvone using Phoma sp., a filamentous fungus from human skin. Biocatalysis provides some advantages, such as high efficiency, high degrees of regioselectivity, chemoselectivity, and enantioselectivity. In order to optimize the small scale biocatalytic reaction of the (4S)-(+)-carvone by the filamentous fungus Phoma sp. was used headspace GC/MS methodology, factorial design of experiments and the response surface methodology (RSM) was performed using the biomass of the fungus, substrate mass and pH as parameters. It was observed that for all reactions conditions tested, forming the products (1 R,4S)-dihydrocarvone and (1S,4S)-dihydrocarvone. The most influential factor was pH, with the highest conversion rate (>95%) and diastereomeric excess (d.e.) (>80%) obtained at pH 5.0. Thus, it was demonstrated that human skin Phoma sp. fungus showed significant bioreduction activity and that headspace GC/MS is an efficient approach for real-time monitoring the biocatalysed reactions. [ABSTRACT FROM AUTHOR]

Ene‐reductases from the old yellow enzyme (OYE) family have been traditionally employed in the reduction of conjugated C═C double bonds. This study explores the underutilized oxidative potential of OYEs, demonstrating their capability to catalyze the enantioselective desaturation of carbonyl compounds. Utilizing a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE‐catalyzed desaturation at ambient temperature and alkaline pH without the need for high‐temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral α,β‐unsaturated carbonyl compounds. [ABSTRACT FROM AUTHOR]

12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer–dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis. [ABSTRACT FROM AUTHOR]

Enzymes capable of assimilating fluorinated feedstocks are scarce. This situation poses a challenge for the biosynthesis of fluorinated compounds used in pharmaceuticals, agrochemicals, and materials. We developed a photoenzymatic hydrofluoroalkylation that integrates fluorinated motifs into olefins. The photoinduced promiscuity of flavin-dependent ene-reductases enables the generation of carbon-centered radicals from iodinated fluoroalkanes, which are directed by the photoenzyme to engage enantioselectively with olefins. This approach facilitates stereocontrol through interaction between a singular fluorinated unit and the enzyme, securing high enantioselectivity at β, γ, or δ positions of fluorinated groups through enzymatic hydrogen atom transfer—a process that is notably challenging with conventional chemocatalysis. This work advances enzymatic strategies for integrating fluorinated chemical feedstocks and opens avenues for asymmetric synthesis of fluorinated compounds. [ABSTRACT FROM AUTHOR]

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of C C bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving C C bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems. [Display omitted] • C C reduction is a key transformation in the synthesis of flavours and fragrances. • Biocatalytic alternatives involve ene-reductases or microorganisms habouring them. • Many examples of industrially relevant molecules are provided and discussed. • Multi-enzymatic systems involving C C bioreductions are also examined. [ABSTRACT FROM AUTHOR]

The Cover shows the one‐pot coupling of ene‐reductases and ω‐transaminases to achieve the biocatalytic synthesis of diastereomerically enriched (R)‐ and (S)‐amines containing an additional stereocenter starting from either α‐ or β‐substituted unsaturated ketones as substrates. In their Communication, S. Riva and co‐workers demonstrate that the amine products can be obtained with excellent stereoselectivity and up to quantitative conversion in sequential and even cascade processes, with no need to adapt the reaction conditions to the reductive amination step or to purify the reaction intermediates. More details can be found in the Communication by S. Riva et al. on page 3106 in Issue 19, 2015 (DOI: 10.1002/cctc.201500424). [ABSTRACT FROM AUTHOR]