Ene Reductase Research Articles

Identification of Five Robust Novel Ene-Reductases from Thermophilic Fungi

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.

Asymmetric Monoreduction of α,β-Dicarbonyls to α-Hydroxy Carbonyls by Ene Reductases.

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

One-Pot Multienzyme Cascades for Stereodivergent Synthesis of Tetrahydroquinolines.

The tetrahydroquinoline (THQ) framework is commonly found in natural products and pharmaceutically relevant molecules. Apart from the use of transition metal catalysts and chiral phosphoric acids, the chiral 2-substituted 1,2,3,4-THQs are synthesized using amine oxidase biocatalysts. However, the use of imine reductases (IREDs) in their asymmetric synthesis remained unexplored. In the current work, IREDs are employed in telescopic multienzyme cascades to catalyze the intramolecular reductive amination leading to chiral 2-alkyl and 2-aryl substituted-1,2,3,4-tetrahydroquinolines starting from inexpensive nitroalkenones. The cascades containing NtDBR (an ene reductase), NfsB (a nitro reductase) with either Na2S2O4 or V2O5, various IREDs, and glucose dehydrogenase (for NADPH regeneration) are used to synthesize a broad range of (R)/(S)-2-alkyl-substituted (THQs) (26 examples) with high yield (up to 93%) and excellent ee (up to 99%) in one-pot. The method further facilitates the one-pot biocatalytic synthesis of chiral 2-aryl substituted THQs (26 examples) from amino chalcones. Lastly, the asymmetric synthesis of several (R)- and (S)-THQ based intermediates of Hancock alkaloids showed the practical application of the newly developed biocatalytic cascades.

Applications of Ene-Reductases in the Synthesis of Flavors and Fragrances.

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

Immobilised Alcohol Dehydrogenase and Ene‐Reductase Work in Concert to Reduce Cyclohex‐2‐enone in Bulk Organic Solvent

Enone reduction by ene reductase (ERED) has not been reported yet in bulk organic media probably due to expected challenges with the cofactor recycling using a second enzyme and the exchange of the NAD(P)‐cofactor between the two enzymes. Herein, the combination of an ERED from Zymomonas mobilis (NCR‐ERED) and Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) has been found as an efficient biocatalytic redox system for the reduction of cyclohex‐2‐enone into cyclohexanol in bulk organic solvents. Both enzymes were successfully immobilised on three EziG supports, finding Coral as the most promising carrier for both of them. The highest enzymatic activities were found in hydrophobic solvents such as toluene and isooctane. It was observed that the control of water content and post‐immobilisation treatments enabled retention of enzyme activity. Both enzymes were co‐immobilised on the same bead together with NADP+, leading to the production of a self‐sufficient catalytic system, although the external supplementation of the nicotinamide cofactor was necessary for an efficient enzyme reuse. In this case, no ERED activity loss was detected after two additional cycles, while the ADH experienced less than 20% activity loss upon reuse.

Solvent concentration at 50% protein unfolding may reform enzyme stability ranking and process window identification

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 cU50T\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${c}_{{U}_{50}}^{T}$$\\end{document} is introduced. Analyzing a set of ene reductases, cU50T\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${c}_{{U}_{50}}^{T}$$\\end{document} 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 cU50T\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${c}_{{U}_{50}}^{T}$$\\end{document} reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of cU50\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${c}_{{U}_{50}}$$\\end{document} versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

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

Peroxygenase-Catalyzed Allylic Oxidation Unlocks Telescoped Synthesis of (1S,3R)-3-Hydroxycyclohexanecarbonitrile.

The unmatched chemo-, regio-, and stereoselectivity of enzymes renders them powerful catalysts in the synthesis of chiral active pharmaceutical ingredients (APIs). Inspired by the discovery route toward the LPA1-antagonist BMS-986278, access to the API building block (1S,3R)-3-hydroxycyclohexanecarbonitrile was envisaged using an ene reductase (ER) and alcohol dehydrogenase (ADH) to set both stereocenters. Starting from the commercially available cyclohexene-1-nitrile, a C-H oxyfunctionalization step was required to introduce the ketone functional group, yet several chemical allylic oxidation strategies proved unsuccessful. Enzymatic strategies for allylic oxidation are underdeveloped, with few examples on selected substrates with cytochrome P450s and unspecific peroxygenases (UPOs). In this case, UPOs were found to catalyze the desired allylic oxidation with high chemo- and regioselectivity, at substrate loadings of up to 200 mM, without the addition of organic cosolvents, thus enabling the subsequent ER and ADH steps in a three-step one-pot cascade. UPOs even displayed unreported enantioselective oxyfunctionalization and overoxidation of the substituted cyclohexene. After screening of enzyme panels, the final product was obtained at titers of 85% with 97% ee and 99% de, with a substrate loading of 50 mM, the ER being the limiting step. This synthetic approach provides the first example of a three-step, one-pot UPO-ER-ADH cascade and highlights the potential for UPOs to catalyze diverse enantioselective allylic hydroxylations and oxidations that are otherwise difficult to achieve.

Regiodivergent Radical Termination for Intermolecular Biocatalytic C-C Bond Formation.

Radical hydrofunctionalizations of electronically unbiased dienes are challenging to render regioselective, because the products are nearly identical in energy. Here, we report two engineered FMN-dependent "ene"-reductases (EREDs) that catalyze regiodivergent hydroalkylations of cyclic and linear dienes. While previous studies focused exclusively on the stereoselectivity of alkene hydroalkylation, this work highlights that EREDs can control the regioselectivity of hydrogen atom transfer, providing a method for selectively preparing constitutional isomers that would be challenging to prepare using traditional synthetic methods. Engineering the ERED from Gluconabacter sp. (GluER) furnished a variant that favors the γ,δ-unsaturated ketone, while an engineered variant from a commercial ERED panel favors the δ,ε-unsaturated ketone. The effect of beneficial mutations has been investigated using substrate docking studies and the mechanism probed by isotope labeling experiments. A variety of α-bromo ketones can be coupled with cyclic and linear dienes. These interesting building blocks can also be further modified to generate difficult-to-access heterocyclic compounds.

Asymmetric α-benzylation of cyclic ketones enabled by concurrent chemical aldol condensation and biocatalytic reduction

Chemoenzymatic cascade catalysis has emerged as a revolutionary tool for streamlining traditional retrosynthetic disconnections, creating new possibilities for the asymmetric synthesis of valuable chiral compounds. Here we construct a one-pot concurrent chemoenzymatic cascade by integrating organobismuth-catalyzed aldol condensation with ene-reductase (ER)-catalyzed enantioselective reduction, enabling the formal asymmetric α-benzylation of cyclic ketones. To achieve this, we develop a pair of enantiocomplementary ERs capable of reducing α-arylidene cyclic ketones, lactams, and lactones. Our engineered mutants exhibit significantly higher activity, up to 37-fold, and broader substrate specificity compared to the parent enzyme. The key to success is due to the well-tuned hydride attack distance/angle and, more importantly, to the synergistic proton-delivery triade of Tyr28-Tyr69-Tyr169. Molecular docking and density functional theory (DFT) studies provide important insights into the bioreduction mechanisms. Furthermore, we demonstrate the synthetic utility of the best mutants in the asymmetric synthesis of several key chiral synthons.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

Continuous-flow synthesis of polysubstituted γ-butyrolactones via enzymatic cascade catalysis

Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone (3S,4S)-4a and whisky lactone (3R,4S)-4b from α,β-unsaturated γ-ketoesters. A new ene reductase (ER) from Swingsia samuiensi (SsER) and a carbonyl reductase (SsCR) were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating (3S,4S)-4a (99% ee and 87% de) and (3R,4S)-4b (99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.

Regioselective Radical Alkylation of Arenes Using Evolved Photoenzymes.

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. Regioselective C-H functionalization reactions are attractive for preparing alkylated arenes; however, the selectivity of existing methods is modest and primarily governed by the substrate's electronic properties. Here, we demonstrate a biocatalyst-controlled method for the regioselective alkylation of electron-rich and electron-deficient heteroarenes. Starting from an unselective "ene"-reductase (ERED) (GluER-T36A), we evolved a variant that selectively alkylates the C4 position of indole, an elusive position using prior technologies. Mechanistic studies across the evolutionary series indicate that changes to the protein active site alter the electronic character of the charge transfer (CT) complex responsible for radical formation. This resulted in a variant with a significant degree of ground-state CT in the CT complex. Mechanistic studies on a C2-selective ERED suggest that the evolution of GluER-T36A helps disfavor a competing mechanistic pathway. Additional protein engineering campaigns were carried out for a C8-selective quinoline alkylation. This study highlights the opportunity to use enzymes for regioselective radical reactions, where small molecule catalysts struggle to alter selectivity.

Photoenzymatic Hydrosulfonylation for the Stereoselective Synthesis of Chiral Sulfones.

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).

Photoenzymatic Hydrosulfonylation for the Stereoselective Synthesis of Chiral Sulfones

AbstractChiral 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).

Ene Reductase Enabled Intramolecular β-C-H Functionalization of Substituted Cyclohexanones for Efficient Synthesis of Bridged Bicyclic Nitrogen Scaffolds.

Herein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular β-C-H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6-azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram-scale one-pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N-phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6-azabicyclo[3.2.1]octan-3-one into 6-azabicyclo[3.2.1]octan-3α-ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3-substituted cyclohexanone derivatives to form the α,β-unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza-Michael addition under basic conditions.

Ene Reductase Enabled Intramolecular β‐C−H Functionalization of Substituted Cyclohexanones for Efficient Synthesis of Bridged Bicyclic Nitrogen Scaffolds

AbstractHerein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular β‐C−H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6‐azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram‐scale one‐pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N‐phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6‐azabicyclo[3.2.1]octan‐3‐one into 6‐azabicyclo[3.2.1]octan‐3α‐ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3‐substituted cyclohexanone derivatives to form the α,β‐unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza‐Michael addition under basic conditions.

Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,β-Unsaturated Aldehydes in Listeria monocytogenes.

The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,β-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,β-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,β-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field.

Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

Asymmetric C-Alkylation of Nitroalkanes via Enzymatic Photoredox Catalysis.

Tertiary nitroalkanes and the corresponding α-tertiary amines represent important motifs in bioactive molecules and natural products. The C-alkylation of secondary nitroalkanes with electrophiles is a straightforward strategy for constructing tertiary nitroalkanes; however, controlling the stereoselectivity of this type of reaction remains challenging. Here, we report a highly chemo- and stereoselective C-alkylation of nitroalkanes with alkyl halides catalyzed by an engineered flavin-dependent "ene"-reductase (ERED). Directed evolution of the old yellow enzyme from Geobacillus kaustophilus provided a triple mutant, GkOYE-G7, capable of synthesizing tertiary nitroalkanes in high yield and enantioselectivity. Mechanistic studies indicate that the excitation of an enzyme-templated charge-transfer complex formed between the substrates and cofactor is responsible for radical initiation. Moreover, a single-enzyme two-mechanism cascade reaction was developed to prepare tertiary nitroalkanes from simple nitroalkenes, highlighting the potential to use one enzyme for two mechanistically distinct reactions.