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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]

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 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]

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]

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]

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]

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]

• 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]

(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]

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 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.

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short c U 50 T is introduced. Analyzing a set of ene reductases, c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature. Melting temperature is the parameter generally used as a measure for stability of enzymes. Here, the authors show that that the melting temperature does not necessarily correlate to the activity observed in the presence of the solvent, and introduce an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T. [ABSTRACT FROM AUTHOR]

Chiral sulfones are recurrent motifs in pharmaceuticals and bioactive molecules. Although chemical methods have been developed to afford α‐ or β‐ chiral sulfones, these protocols rely heavily on the pre‐synthesis of structurally complicated starting materials and chiral metal complexes. Herein, we described a photoenzymatic approach for the radical‐mediated stereoselective hydrosulfonylation. Engineered variants of ene reductases provide efficient biocatalysts for this transformation, enabling to achieve a series of β‐chiral sulfonyl compounds with high yields (up to 92 %) and excellent e.r. values (up to 99 : 1). [ABSTRACT FROM AUTHOR]

Reductive radical cyclizations are ubiquitous in organic synthesis and have been applied to the synthesis of structurally complex molecules. N-Heterocyclic motifs can be prepared through the cyclization of α-haloamides; however, slow rotation around the amide C–N bond results in preferential formation of an acyclic hydrodehalogenated product. Here, we compare four different methods for preparing γ-, δ-, ε-, and ζ-lactams via radical cyclization. We found that a photoenzymatic method using flavin-dependent 'ene' reductases affords the highest level of product selectivity. We suggest that through selective binding of the cis -amide isomer, the enzyme preorganizes the substrate for cyclization, helping to avoid premature radical termination. [ABSTRACT FROM AUTHOR]

[Display omitted] • Identification of the key residues for the stereo-control of ene reductase OYE1. • Engineering of OYE1 for the synthesis of active (S)-profen derivatives. • Up to >99 % stereoselectivity was observed for the asymmetric reduction. 2-Arylpropionic acid derivatives, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of 2-arylacrylic acid derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction by EREDs resulted solely in nonpharmacological (R)-enantiomers. Here, we present the engineering of Saccharomyces pastorianus old yellow enzyme 1 (OYE1) for the synthesis of pharmacologically active (S)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro- S model of OYE1, the key residues of OYE1 were identified. Mutagenesis screening at these positions successfully provided the (S)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (S)-derivatives with up to >99 % ee values. Moreover, the key residues obtained from OYE1 successfully guided the engineering of OYE2.6 from Pichia stipites and KnOYE1 from Kazachstania naganishii for (S)-selective reduction of 2-arylacrylic acid derivatives. [ABSTRACT FROM AUTHOR]

Access to chiral amines with more than one stereocentre remains challenging, although an increasing number of methods are emerging. Here we developed a proof‐of‐concept bi‐enzymatic cascade, consisting of an ene reductase and amine dehydrogenase (AmDH), to afford chiral diastereomerically enriched amines in one pot. The asymmetric reduction of unsaturated ketones and aldehydes by ene reductases from the Old Yellow Enzyme family (OYE) was adapted to reaction conditions for the reductive amination by amine dehydrogenases. By studying the substrate profiles of both reported biocatalysts, thirteen unsaturated carbonyl substrates were assayed against the best duo OYE/AmDH. Low (5 %) to high (97 %) conversion rates were obtained with enantiomeric and diastereomeric excess of up to 99 %. We expect our established bi‐enzymatic cascade to allow access to chiral amines with both high enantiomeric and diastereomeric excess from varying alkene substrates depending on the combination of enzymes. [ABSTRACT FROM AUTHOR]

Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone. [ABSTRACT FROM AUTHOR]

The asymmetric bioreduction of prochiral conjugated alkenes using ene-reductases allows powerful strategies to access both enantiomers of the product with high stereoselectivity. This may be achieved (i) by using pairs of (iso)enzymes, which bind the alkene moiety in mirror-image orientations to affect hydride attack from opposite sides, (ii) via a switch in the (E/Z)-geometry of the alkene unit, or (iii) by changing the size of the protective groups of the substrate, which enforces a flipped orientation in the active site. Modeling studies provide a rationale for the molecular basis of substrate binding and allow the prediction of the stereochemical outcome of this useful bioreduction. 1 Introduction 2 Enzyme-Based Stereocontrol: 'Enantiomeric' Ene-reductases 3 Substrate-Based Stereocontrol: Flipping Substrates 3.1 Stereocontrol via (E/Z)-Configuration of Substrate 3.2 Stereocontrol via Substituent Effects 4 Modeling of Substrate Complexes 5 Conclusions [ABSTRACT FROM AUTHOR]

• A bi-functional redox biocatalyst was obtained by co-immobilization of ene-reductase and glucose dehydrogenase. • The bi-functional biocatalyst showed increased conversion compared to the combined use of individual catalysts. • Excellent reusability of the co-immobilized biocatalyst was shown over 10 cycles. • Preparative-scale synthesis allowed access to important chemical building blocks in high enantiopurity. An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions. [ABSTRACT FROM AUTHOR]

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N-substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N-substituted 4-hydroxy-phenyl residue led to a sixfold higher vmax relative to the natural cofactor NADH. [ABSTRACT FROM AUTHOR]

Ene reductases (EREDs) have great potential as oxidation biocatalysts, as demonstrated by their efficient conversion of a number of tetralones to the corresponding naphthols. Of 96 enzymes tested, 57 were able to produce 2-naphthol in this way. Further tests with substituted tetralones revealed typically high conversions up to >99%. The reactions were performed under mild conditions in aqueous buffer with only co-solvent, biocatalyst and oxidation substrate required for conversion. Production of a methoxy-substituted naphthol was also successfully performed on a gram scale, with 91% yield. This methodology provides a new avenue to produce substituted naphthols as valuable building blocks, with the possibility to extend the approach to the production of phenols also being demonstrated. [ABSTRACT FROM AUTHOR]

The reduction of ( Z)-β-acylaminonitroalkenes catalyzed by ene-reductases is described for the first time. The reaction occurs with a high conversion and excellent enantioselectivity and shows a wide substrate scope. The reduced products are valuable chiral synthons characterized by two vicinal nitrogen-containing functional groups that can be further modified by functional group inter-conversion thanks to the synthetic versatility of the nitro moiety. The chemo-enzymatic synthesis of ( R)- N, N′-(1-phenylethane-1,2-diyl)diacetamide from easily accessible ( Z)- N-(2-nitro-1-phenylvinyl)acetamide is herein reported as a representative application of this synthetic procedure. [ABSTRACT FROM AUTHOR]

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)-menthone:(−)-menthol reductase and (−)-menthone:(+)-neomenthol reductase, and the 'ene' reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. [ABSTRACT FROM AUTHOR]

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of 'thermophilic-like' OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses noncovalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37℃. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32℃ from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed. [ABSTRACT FROM AUTHOR]

Ene reductases from the Old Yellow Enzyme family are versatile biocatalysts useful for the synthesis of optically active compounds. One disadvantage of biocatalysts when compared to competing catalysts in chemical syntheses is that often only one stereoisomer of the product is available. Another drawback can be the lack of activity in certain enzyme-substrate combinations. We were able to approach both of these challenges rationally in the case of the enzymatic synthesis of methyl 3-hydroxy-2-methylpropanoate (commonly denoted as the Roche ester) and derivatives thereof using the ene reductase YqjM. By a highly efficient, concept-based approach of designing mutant variants of YqjM and engineering substrates we could alter both the rate constant and the enantioselectivity of the reaction. Preparative scale reactions have been performed with successful mutants. In addition, the iterative modification of the substrate gave experiment-based insights into the binding mode of the Roche ester precursor and its derivatives. [ABSTRACT FROM AUTHOR]

Chiral amines are pivotal building blocks for the pharmaceutical industry. Asymmetric reductive amination is one of the most efficient and atom economic methodologies for the synthesis of optically active amines. Among the various strategies available, NAD(P)H-dependent amine dehydrogenases (AmDHs) and imine reductases (IREDs) are robust enzymes that are available from various sources and capable of utilizing a broad range of substrates with high activities and stereoselectivities. AmDHs and IREDs operate via similar mechanisms, both involving a carbinolamine intermediate followed by hydride transfer from the co-factor. In addition, both groups catalyze the formation of primary and secondary amines utilizing both organic and inorganic amine donors. In this review, we discuss advances in developing AmDHs and IREDs as biocatalysts and focus on evolutionary history, substrate scope and applications of the enzymes to provide an outlook on emerging industrial biotechnologies of chiral amine production. [ABSTRACT FROM AUTHOR]

Biocatalysis has emerged as a valuable and reliable tool for industrial and academic societies, particularly in fields related to bioredox reactions. The cost of cofactors, especially those needed to be replenished at stoichiometric amounts or more, is the chief economic concern for bioredox reactions. In this study, a readily accessible, inexpensive, and bench‐stable Hantzsch ester is verified as the viable and efficient NAD(P)H mimic by four enzymatic redox transformations, including two non‐heme diiron N‐oxygenases and two flavin‐dependent reductases. This finding provides the potential to significantly reduce the costs of NAD(P)H‐relying bioredox reactions. [ABSTRACT FROM AUTHOR]

Chiral β‐aryl propanamine is an important structure unit of bioactive molecules or drugs. But its efficient synthesis remains a challenging task. In this study, several enantiocomplementary imine reductases capable of dynamic kinetic resolution‐reductive amination of sixteen 2‐phenylpropanal derivatives of diverse structural characteristics with four different amines were identified through screening 196 imine reductases. Furthermore, (S)‐IR60, (R)‐IR74 and (R)‐IR207 were applied to access a series of different β‐aryl propanamines with excellent ee values (90 to 99 %) and high isolated yields (36 to 79 %), and two of them were further transformed into 3‐methylindolines through intramolecular Buchwald‐Hartwig cross‐coupling and deallylation, providing an effective method to construct this class of pharmaceutically important compounds. [ABSTRACT FROM AUTHOR]

While many redox enzymes are nowadays available for synthetic applications, the toolbox of ene-reductases is still limited. Consequently, the screening for these enzymes from diverse sources in the search of new biocatalyst suitable for green chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were selected due to their ability to hydrogenate the C C double bond of the model substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more preference toward highly activated molecules such as nitrostyrene and maleimide rather than the classical substrates of the well-known Old Yellow Enzymes, resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When the three biocatalytic systems were applied in the reduction of chalcones, T. absinthioides showed high chemoselectivity toward the C C double bond whereas the other two demonstrated abilities to biohydrogenate the C C double bounds and the carbonyl groups in a sequential fashion. [ABSTRACT FROM AUTHOR]

Two putative Old Yellow Enzyme (OYE) homologues, Chr -OYE1 and Chr -OYE2, were identified from the genome of Chryseobacterium sp. CA49 as new members of the “classical” subfamily. Chr -OYE1 and Chr -OYE2 were most closely related to the SYE4 from Shewanella oneidensis and NerA from Agrobacterium radiobacter with 41% and 45% identity, respectively. Both enzymes were expressed in Escherichia coli in soluble form, but their catalytic abilities as ene-reductases were quite different. Among the 19 substrate tested, Chr -OYE1 could catalyze the reduction of 18 of them including an ynone with excellent stereoselectivity for several prochiral ones, and its specific activity was roughly 1100-fold high than Chr -OYE2, which only catalyzed 3 of the substrates. After restoring the conserved tyrosine, Chr -OYE2 remained the same substrate spectrum, but showed significantly enhanced activity and stereoselectivity. [ABSTRACT FROM AUTHOR]

Biotransformations were performed on eight selected yeast strains, all of which were able to selectively hydrogenate the chalcone derivatives 3-(2"-furyl)- (1) and 3-(2"-thienyl)-1-(2'-hydroxyphenyl)-prop-2-en-1-one (3) into 3-(2"-furyl)- (2) and 3-(2"-thienyl)-1-(2'-hydroxyphenyl)-propan-1-one (4) respectively. The highest efficiency of hydrogenation of the double bond in the substrate 1 was observed in the cultures of Saccharomyces cerevisiae KCh 464 and Yarrowia lipolytica KCh 71 strains. The substrate was converted into the product with > 99% conversion just in six hours after biotransformation started. The compound containing the sulfur atom in its structure was most effectively transformed by the Yarrowia lipolytica KCh 71 culture strain (conversion > 99%, obtained after three hours of substrate incubation). Also, we observed that, different strains of tested yeasts are able to carry out the bioreduction of the used substrate with different yields, depending on the presence of induced and constitutive ene reductases in their cells. The biggest advantage of this process is the efficient production of one product, practically without the formation of side products. [ABSTRACT FROM AUTHOR]

The development of mild, efficient, and enantioselective methods for preparing chiral fluorinated compounds has been a long‐standing challenge. Herein, we report a promiscuous cyclohexanone monooxygenase (CHMO) for the photoinduced synthesis of chiral α‐fluoroketones via enantioselective reductive dehalogenation of α,α‐halofluoroketones. Wild‐type CHMO from Acinetobacter sp. possesses this promiscuous ability innately; however, the yield and stereoselectivity are low. A structure‐guided rational design of CHMO improved the yield and stereoselectivity remarkably. Mechanistic studies and molecular simulations demonstrated that this photoinduced CHMO catalyzes the reductive dehalogenation via a novel electron transfer (ET)/proton transfer (PT) mechanism, distinct from that of previously reported reductases with similar promiscuity. This methodology was expanded to various substrates, and desirable chiral α‐fluoroketones were obtained in high yields (up to 99 %) and e.r. values (up to 99:1). [ABSTRACT FROM AUTHOR]

The synthesis of chiral amines is significant in the pharmaceutical industry. Imine reductase (IRED), a promising biocatalyst that was previously known to only catalyze asymmetric imine reduction (IR), was revealed to achieve direct asymmetric reductive amination (RA) of ketones with excess amines, producing secondary and tertiary amines. Moreover, conjugate reduction (CR) and RA activity by a single IRED has been reported for the preparation of valuable amine diastereomers. IREDs catalyzing these different reactions share the same standard quaternary structure, but possess varying active sites, indicating correlations and differences between their catalytic mechanisms. In this review, we trace the catalytic mechanisms reported for IRED‐catalyzed IR, RA, and CR‐RA. Insights obtained from structural and protein engineering in understanding the IRED‐catalyzed asymmetric synthesis are also included. This review will help readers acquire comprehensive insights into the catalytic mechanism of IREDs and ultimately inspire the engineering of IREDs for industrial applications. [ABSTRACT FROM AUTHOR]

The bioreduction and disproportionation of cyclohex-2-enone catalyzed by Old Yellow Enzyme 1 was investigated in presence of organic (co)solvents. Whereas the NADH-dependent bioreduction activity strongly decreased at elevated co-solvent concentrations due to the insolubility of the nicotinamide-cofactor, the NADH-free disproportionation was significantly improved in water-immiscible organic co-solvents at 90 % (v/v) with near-quantitative conversion. This positive effect was attributed to removal of the inhibiting co-product, phenol, from the enzyme's active site. The best co-solvents show high lipophilicity (logP) and a high potential to solubilize phenol (K). As a predictive parameter, the ratio of logP/K should be preferably ≥100. [ABSTRACT FROM AUTHOR]

The concurrent operation of chemical and biocatalytic reactions in one pot is still a challenging task, and, in particular for chemical photocatalysts, examples besides simple cofactor recycling systems are rare. However, especially due to the complementary chemistry that the two fields of catalysis promote, their combination in one pot has the potential to unlock intriguing, unprecedented overall reactivities. Herein we demonstrate a concurrent biocatalytic reduction and photocatalytic oxidation process. Specifically, the enantioselective biocatalytic sulfoxide reduction using (S)‐selective methionine sulfoxide reductases was coupled to an unselective light‐dependent sulfoxidation. Protochlorophyllide was established as a new green photocatalyst for the sulfoxidation. Overall, a cyclic deracemization process to produce nonracemic sulfoxides was achieved and the target compounds were obtained with excellent conversions (up to 91 %) and superb optical purity (>99 % ee). [ABSTRACT FROM AUTHOR]

2‐Aminotetralin and 3‐aminochroman derivatives are key structural motifs present in a wide range of pharmaceutically important molecules. Herein, we report an effective biocatalytic approach towards these molecules through the enantioselective reductive coupling of 2‐tetralones and 3‐chromanones with a diverse range of primary amine partners. Metagenomic imine reductases (IREDs) were employed as the biocatalysts, obtaining high yields and enantiocomplementary selectivity for >15 examples at preparative scale, including the precursors to Ebalzotan, Robalzotan, Alnespirone and 5‐OH‐DPAT. We also present a convergent chemo‐enzymatic total synthesis of the Parkinson's disease therapy Rotigotine in 63 % overall yield and 92 % ee. [ABSTRACT FROM AUTHOR]

Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR‐17, when co‐expressed with a co‐factor recycling system produced the anti‐(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis. [ABSTRACT FROM AUTHOR]

This study introduces a new type of ene/yne‐reductase from Cyclocybe aegerita with a broad substrate scope including aliphatic and aromatic alkenes/alkynes from which aliphatic C8‐alkenones, C8‐alkenals and aromatic nitroalkenes were the preferred substrates. By comparing alkenes and alkynes, a ∼2‐fold lower conversion towards alkynes was observed. Furthermore, it could be shown that the alkyne reduction proceeds via a slow reduction of the alkyne to the alkene followed by a rapid reduction to the corresponding alkane. An accumulation of the alkene was not observed. Moreover, a regioselective reduction of the double bond in α,β‐position of α,β,γ,δ‐unsaturated alkenals took place. This as well as the first biocatalytic reduction of different aliphatic and aromatic alkynes to alkanes underlines the novelty of this biocatalyst. Thus with this study on the new ene‐reductase CaeEnR1, a promising substrate scope is disclosed that describes conceivably a broad occurrence of such reactions within the chemical landscape. [ABSTRACT FROM AUTHOR]

[Display omitted] • Panel of Ts ER variants for stereocomplementary asymmetric hydrogenation of activated alkenes. • Reaction system stable at elevated temperatures of up to 70 °C. • Organic solvent tolerance up to 40 % v/v. • Bi-phasic systems improved enantiopurity, simplified downstream processes and allowed biocatalysts to be reused. We report an engineered panel of ene-reductases (ERs) from Thermus scotoductus SA-01 (Ts ER) that combines control over facial selectivity in the reduction of electron deficient C C double bonds with thermostability (up to 70 °C), organic solvent tolerance (up to 40 % v/v) and a broad substrate scope (23 compounds, three new to literature). Substrate acceptance and facial selectivity of 3-methylcyclohexenone was rationalized by crystallisation of Ts ER C25D/I67T and in silico docking. The Ts ER variant panel shows excellent enantiomeric excess (ee) and yields during bi-phasic preparative scale synthesis, with isolated yield of up to 93 % for 2 R ,5 S -dihydrocarvone (3.6 g). Turnover frequencies (TOF) of approximately 40 000 h−1 were achieved, which are comparable to rates in hetero- and homogeneous metal catalysed hydrogenations. Preliminary batch reactions also demonstrated the reusability of the reaction system by consecutively removing the organic phase (n -pentane) for product removal and replacing with fresh substrate. Four consecutive batches yielded ca. 27 g L−1 R -levodione from a 45 mL aqueous reaction, containing less than 17 mg (10 μM) enzyme and the reaction only stopping because of acidification. The Ts ER variant panel provides a robust, highly active and stereocomplementary base for further exploitation as a tool in preparative organic synthesis. [ABSTRACT FROM AUTHOR]

Reductions play a key role in organic synthesis, producing chiral products with new functionalities. Enzymes can catalyse such reactions with exquisite stereo‐, regio‐ and chemoselectivity, leading the way to alternative shorter classical synthetic routes towards not only high‐added‐value compounds but also bulk chemicals. In this review we describe the synthetic state‐of‐the‐art and potential of enzymes that catalyse reductions, ranging from carbonyl, enone and aromatic reductions to reductive aminations. [ABSTRACT FROM AUTHOR]

The soluble hydrogenase from Ralstonia eutropha provides an atom efficient regeneration system for reduced flavin cofactors using H2 as an electron source. We demonstrated this system for highly selective ene-reductase-catalyzed C＝C-double bond reductions and monooxygenase-catalyzed epoxidation. Reactions were expanded to aerobic conditions to supply H2O2 for peroxygenase-catalyzed hydroxylations. [ABSTRACT FROM AUTHOR]

Class I benzoyl‐CoA reductases (BCRs) are oxygen‐sensitive key enzymes in the degradation of monocyclic aromatic compounds in anaerobic prokaryotes. They catalyze the ATP‐dependent reductive dearomatization of their substrate to cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA (1,5‐dienoyl‐CoA). An aromatizing 1,5‐dienoyl‐CoA oxidase (DCO) activity has been proposed to protect BCRs from oxidative damage, however, the gene and its product involved have not been identified, yet. Here, we heterologously produced a DCO from the hyperthermophilic euryarchaeon Ferroglobus placidus that coupled the oxidation of two 1,5‐dienoyl‐CoA to benzoyl‐CoA to the reduction of O2 to water at 80°C. DCO showed similarities to members of the old yellow enzyme family and contained FMN, FAD and an FeS cluster as cofactors. The O2‐dependent activation of inactive, reduced DCO is assigned to a redox thiol switch at Eo′ = −3 mV. We propose a catalytic cycle in which the active site FMN/disulfide redox centers are reduced by two 1,5‐dienoyl‐CoA (reductive half‐cycle), followed by two consecutive two‐electron transfer steps to molecular oxygen via peroxy‐ and hydroxyflavin intermediates yielding water (oxidative half‐cycle). This work identified the enzyme involved in a unique oxygen detoxification process for an oxygen‐sensitive catabolic enzyme. [ABSTRACT FROM AUTHOR]

This study applied the use of marine-derived fungus Penicillium citrinum CBMAI 1186 in the stereoselective reduction of the C=C double bond of the prochiral (E)-2-methyl-3-phenylacrylaldehyde 1. The fungus immobilized on chitosan, obtained by multistep ultrasound-assisted deacetylation process (Ch-USAD), produced the (S)-(+)-2-methyl-3-phenylpropan-1-ol 3 (c = 49%, 40% ee) isomer and (±)-2-methyl-3-phenylacrilic acid 4 (c = 35%); in contrast, immobilized mycelia on commercial chitosan (Ch-C) yielded the (S)-(+)-2-methyl-3-phenylpropan-1-ol 3 (c = 48%, 10% ee) and (±)-2-methyl-3-phenylpropanal 1a (c = 41%). The reaction using free mycelia gave a 40% yield of (S)-(+)-2-methyl-3-phenylpropan-1-ol 3 with 10% ee. These results showed that the crystallinity form and molecular weight of chitosan (Ch-C or Ch-USAD) used to immobilized mycelia of P. citrinum CBMAI 1186 influenced in the biotransformation of (E)-2-methyl-3-phenylacrylaldehyde 1. Therefore, marine-derived fungus P. citrinum CBMAI 1186 immobilized on chitosan can be a potential alternative in the studies of hydrogenation of the α,β-unsaturated carbon-carbon (α,β-C=C) double bond. [ABSTRACT FROM AUTHOR]

Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (2H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2H]-NADH). Here we report on a strategy that combines a clean reductant (H2) with a cheap source of 2H-atoms (2H2O) to generate and recycle [4-2H]-NADH. By coupling [4-2H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2H3]-solifenacin fumarate on a preparative scale. Deuterated chemicals are widely exploited in analytical chemistry and increasingly in the synthesis of pharmaceutical compounds. Here, the authors developed a mild biocatalytic method for the selective asymmetric reductive deuteration of organic compounds, by using H2 and 2H2O to generate 2H-NADH. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel ene-reductase from Meyerozyma guilliermondii and achieve the ene-reductase-mediated reduction of activated C=C bonds. Results: The gene encoding an ene-reductase was cloned from M. guilliermondii. Sequence homology analysis showed that MgER shared the maximal amino acid sequence identity of 57 % with OYE2.6 from Scheffersomyces stipitis. MgER showed the highest specific activity at 30 °C and pH 7 (100 mM sodium phosphate buffer), and excellent stereoselectivities were achieved for the reduction of ( R)-carvone and ketoisophorone. Under the reaction conditions (30 °C and pH 7.0), 150 mM ( R)-carvone could be completely converted to ( 2R,5R)-dihydrocarvone within 22 h employing purified MgER as catalyst, resulting in a yield of 98.9 % and an optical purity of >99 % d. e. Conclusion: MgER was characterized as a novel ene-reductase from yeast and showed great potential for the asymmetric reduction of activated C=C bonds of α,β-unsaturated compounds. [ABSTRACT FROM AUTHOR]

This study aimed to define progesterone 5β-reductases (P5βR, EC 1.3.99.6, enone 1,4-reductases) as function-associated molecular markers at the plant family level. Therefore cDNAs were isolated from 25 Brassicaceae species, including two species, Erysimum crepidifolium and Draba aizoides, known to produce cardiac glycosides. The sequences were used in a molecular phylogeny study. The cladogram created is congruent to the existing molecular analyses. Recombinant His-tagged forms of the P5β R cDNAs from Aethionema grandiflorum, Draba aizoides, Nasturtium officinale, Raphanus sativus and Sisymbrium officinale were expressed in E. coli. Enone 1,4-reductase activity was demonstrated in vitro using progesterone and 2-cyclohexen-1-one as substrates. Evidence is provided that functional P5βRs are ubiquitous in the Brassicaceae. The recombinant P5βR enzymes showed different substrate preferences towards progesterone and 2-cyclohexen-1-one. Sequence comparison of the catalytic pocket of the P5βR enzymes and homology modelling using Digitalis lanata P5βR ( PDB ID: 2V6G) as template highlighted the importance of the hydrophobicity of the binding pocket for substrate discrimination. It is concluded that P5β R genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective. [ABSTRACT FROM AUTHOR]