RESUMO
Ene-reductase (ER) has been widely applied for asymmetrical synthesis of chiral intermediates due to its substrate promiscuity, photoexcited reactivity, and excellent property with producing two chiral centers at a time. Natural ERs often exhibit the same stereoselectivity, and they need to be engineered for opposite configuration of chiral compounds. The hydrogenation process toward activated alkenes by ERs is composed of reductive half reaction and oxidative half reaction, which are dependent upon two cofactors NAD(P)H and flavin mononucleotide. The catalytic activity of ERs will be affected by the size of the substrate, the activating strength of the electron-withdrawing groups, redox potential of cofactors, and the loop flexibility around catalytic cavity. Currently, protein engineering to ERs has been successfully employed to enhance various catalytic properties, including photoexcited asymmetric synthesis. This review summarizes the approaches to reverse the stereoselectivity and enhance catalytic activity of ERs and new applications of the engineered ERs in photobiocatalytic asymmetric synthesis, besides the discussion with the existing molecular mechanisms of mutants regarding the improved catalytic performance.
RESUMO
Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5-8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, ß-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. KEY POINTS: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2 of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv.
Assuntos
Aspergillus flavus , NADPH Desidrogenase , Aspergillus flavus/metabolismo , NADPH Desidrogenase/metabolismo , Domínio Catalítico , Catálise , AlcenosRESUMO
Direct aromatization of cyclohexanones to synthesize substituted phenols represents a significant challenge in modern synthetic chemistry. Herein, we describe a novel ene-reductase (TsER) catalytic system that converts substituted cyclohexanones into the corresponding phenols. This process involves the successive dehydrogenation of two saturated carbon-carbon bonds within the six-membered ring of cyclohexanones and utilizes molecular oxygen to drive the reaction cycle. It demonstrates a versatile and efficient approach for the synthesis of substituted phenols, providing a valuable complement to existing chemical methodologies.
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(R)-4-Propyldihydrofuran-2(3H)-one (R-PDFO) is the key chiral intermediate for the antiepileptic drug Brivaracetam. Lacking a simple and economical method to approaching R-PDFO, the production of R-PDFO also remains environmentally unfriendly. Here, we developed a straightforward bioreduction way from easily synthesized 4-propylfuran-2(5H)-one (PFO) using ene-reductases. After screened with 27 ene-reductases, E116 stood out with 25.7% yield and 97% ee (R) as the starting enzyme. To improve the catalytic efficiency of E116, several rounds of directed evolution were first carried out. Through rational design, alanine scanning and random mutagenesis, engineered ene-reductase E116-M3 was obtained, with a 2.63-fold improvement in yields over WT, a 12.6-fold improvement in kcat/Km over WT, and stereoselectivity increased to 99% (R). To further improve the yield of R-PDFO, the reaction conditions were then optimized. The catalytic activity of the optimized reaction system was increased again by 2.3 times and the turnover number (TON) of E116-M3 reached 705. Subsequently, whole cells harboring E116-M3 were also shown to have similar capabilities of synthesizing R-PDFO. Finally, E116-M3 was employed in the 50-mL-scale synthesis of R-PDFO under 20 mM of PFO loading to achieve 81% isolated yield and 99% ee. In conclusion, this new approach of engineered ene-reductase catalyzing the asymmetric reduction of PFO could be a green alternative for the efficient synthesis of R-PDFO. KEY POINTS: ⢠An ene-reductase library was first used to screen the bioreduction of PFO. ⢠Rational design contributed to the enhanced R-stereoselectivity of PFO reduction. ⢠E116-M3 was obtained with high activity and stereoselectivity for R-PDFO.
Assuntos
Anticonvulsivantes , Oxirredutases , Oxirredutases/genética , Pirrolidinonas , MutagêneseRESUMO
Asymmetric reduction of (R)-carvone and ketoisophorone by an engineered ene-reductase from Galdieria sulphuraria (GsOYE) combined with glucose dehydrogenase for NADPH regeneration were studied. A semi-rational protein engineering was used to enhance the activity and selectivity of GsOYE. Upon the sequence alignment and molecular docking results, two amino acid residues at positions 66 and 270 were selected as saturation mutation sites. Finally, a single substitution variant of GsOYE-N270A with complete conversion (100%) and diastereoselectivity (dep >99%) for reduction of (R)-carvone and a double substitution variant GsOYE-Y66P/N270H with improved stereoselectivity for reduction of ketoisophorone were obtained.
Assuntos
Oxirredutases , Engenharia de Proteínas , Oxirredutases/metabolismo , Simulação de Acoplamento Molecular , BiocatáliseRESUMO
The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.
Assuntos
Aminoácidos , NADPH Desidrogenase , NADPH Desidrogenase/química , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Monoterpenos Cicloexânicos , Catálise , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
PURPOSE: We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-ß-ionone from ß-ionone. METHODS: KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-ß-ionone from ß-ionone with LC-MS analysis method. RESULTS: The molecular mass of KaDBR1 was estimated to be 45 kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60 °C and pH 6.0. The addition of 5 mM Mg2+, Ca2+, Al3+, Na+, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward ß-ionone using NADH was 8.1-fold greater than when using NADPH. CONCLUSION: Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of ß-ionone to dihydro-ß-ionone in biotechnology applications.
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NAD , Oxirredutases , NADP , Concentração de Íons de Hidrogênio , Peso MolecularRESUMO
To support perfumers in their creation of olfactive signatures resulting in unique and instantly recognizable perfumes, there is a constant demand for the development of new odorant molecules and of novel processes for their production. Increasing the sustainability of both the molecules and the processes is a crucial activity at Givaudan. Biocatalysis has the potential to positively influence metrics applied at Givaudan that drive and measure our ambition to innovate responsibly, which is summarized in the FiveCarbon Path™. It targets an increased use of renewable carbon, carbon efficiency in synthesis, and the production of powerful and biodegradable odorant molecules while maximizing the use of upcycled carbon available from waste and side streams. This review illustrates with some examples how enzymes selected from the oxidoreductase and isomerase enzyme classes are applied at Givaudan for the preparation of odorant molecules both at laboratory and industrial scale.
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Odorantes , Perfumes , Biotecnologia , Biocatálise , Carbono , IndústriasRESUMO
Biocatalysis integrate microbiologists, enzymologists, and organic chemists to access the repertoire of pharmaceutical and agrochemicals with high chemoselectivity, regioselectivity, and enantioselectivity. The saturation of carbon-carbon double bonds by biocatalysts challenges the conventional chemical methodology as it bypasses the use of precious metals (in combination with chiral ligands and molecular hydrogen) or organocatalysts. In this line, Ene-reductases (ERs) from the Old Yellow Enzymes (OYEs) family are found to be a prominent asymmetric biocatalyst that is increasingly used in academia and industries towards unparalleled stereoselective trans-hydrogenations of activated C=C bonds. ERs gained prominence as they were used as individual catalysts, multi-enzyme cascades, and in conjugation with chemical reagents (chemoenzymatic approach). Besides, ERs' participation in the photoelectrochemical and radical-mediated process helps to unlock many scopes outside traditional biocatalysis. These up-and-coming methodologies entice the enzymologists and chemists to explore, expand and harness the chemistries displayed by ERs for industrial settings. Herein, we reviewed the last five year's exploration of organic transformations using ERs.
Assuntos
NADPH Desidrogenase , Oxirredutases , Biocatálise , Carbono , Técnicas de Química Sintética , NADPH Desidrogenase/metabolismo , Oxirredutases/químicaRESUMO
Radical cyclizations are essential reactions in the biosynthesis of secondary metabolites and the chemical synthesis of societally valuable molecules. In this review, we highlight the general mechanisms utilized in biocatalytic radical cyclizations. We specifically highlight cytochrome P450 monooxygenases (P450s) involved in the biosynthesis of mycocyclosin and vancomycin, nonheme iron- and α-ketoglutarate-dependent dioxygenases (Fe/αKGDs) used in the biosynthesis of kainic acid, scopolamine, and isopenicillin N, and radical S-adenosylmethionine (SAM) enzymes that facilitate the biosynthesis of oxetanocin A, menaquinone, and F420. Beyond natural mechanisms, we also examine repurposed flavin-dependent "ene"-reductases (ERED) for non-natural radical cyclization. Overall, these general mechanisms underscore the opportunity for enzymes to augment and enhance the synthesis of complex molecules using radical mechanisms.
Assuntos
Biocatálise , Ciclização , Humanos , Oxirredução , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Vitamina K 2/metabolismoRESUMO
A new activity for the [NiFe] uptake hydrogenaseâ 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 20.4â min-1 and total turnover numbers up to 20 200 were recorded during flavin recycling.
Assuntos
Alcenos/metabolismo , Escherichia coli/enzimologia , Flavinas/metabolismo , Hidrogenase/metabolismo , Oxirredutases/metabolismo , Alcenos/química , Biocatálise , Flavinas/química , Hidrogenase/química , Hidrogenação , Estrutura Molecular , Oxirredução , Oxirredutases/químicaRESUMO
Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) enables the investigation of protein folding in solution. Herein, a proof-of-concept for obtaining structural information about the folding of a protein in dependency of the amount of an organic cosolvent in the aqueous medium by means of this IMS-MS method is presented. By analyzing the protein with native nano-electrospray ionization IMS-MS, the impact of acetonitrile as a representative organic cosolvent and/or pH values on the folding of an enzyme was successfully evaluated in a fast and straightforward fashion, as exemplified for an ene reductase from Gluconobacter oxydans. The IMS-MS results are in agreement with findings from the nicotinamide adenine dinucleotide phosphate (NADPH)-based spectrophotometric enzyme activity tests under analogous conditions, and thus, also rationalizing these "wet" analytical data. For this ene reductase, a higher tolerance against CH3 CN in the presence of a buffer was observed by both analytical methods. The results suggest that this IMS-MS methodology could be a useful complementary tool to existing methods in process optimization and fine-tuning of solvent conditions for biotransformations.
Assuntos
Acetonitrilas/farmacologia , Oxirredutases/metabolismo , Acetonitrilas/química , Estabilidade Enzimática/efeitos dos fármacos , Gluconobacter oxydans/enzimologia , Concentração de Íons de Hidrogênio , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Oxirredutases/análise , Dobramento de Proteína/efeitos dos fármacos , Solventes/química , Solventes/farmacologiaRESUMO
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.
Assuntos
2,2'-Dipiridil/análogos & derivados , Flavoproteínas/química , Compostos Organometálicos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Piridinas/química , Compostos de Vinila/química , 2,2'-Dipiridil/química , 2,2'-Dipiridil/efeitos da radiação , Catálise/efeitos da radiação , Teoria da Densidade Funcional , Hidrogenação , Luz , Modelos Químicos , Nostoc/enzimologia , Compostos Organometálicos/efeitos da radiação , OxirreduçãoRESUMO
(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.
Assuntos
Monoterpenos Cicloexânicos/metabolismo , Oxirredução , Oxirredutases/metabolismo , Biocatálise , Biotransformação , Escherichia coli/metabolismoRESUMO
'Ene'-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The ß,ß-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90â%. By contrast, the α,ß-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56â% and poor enantioselectivity (16â% ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of 'ene'-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds.
Assuntos
Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Bactérias/biossíntese , Biocatálise , Clonagem Molecular , Clostridium/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos/genética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Rhodococcus strains are ubiquitous in nature and known to metabolise a wide variety of compounds. At the same time, asymmetric reduction of C=C bonds is important in the production of high-valued chiral building blocks. In order to evaluate if Rhodococci can be used for this task, we have probed several Rhodococcus rhodochrous and R. erythropolis strains for ene-reductase activity. A series of substrates including activated ketones, an aldehyde, an imide and nitro-compound were screened using whole cells of seven Rhodococcus strains. This revealed that whole cells of all Rhodococcus strains showed apparent (S)-selectivity towards ketoisophorone, while most other organisms show (R)-selectivity for this compound. Three putative ene-reductases from R. rhodochrous ATCC 17895 were heterologously expressed in Escherichia coli. One protein was purified and its biocatalytic and biochemical properties were characterised, showing typical (enantioselective) properties for class 3 ene-reductases of the old yellow enzyme family.
Assuntos
Rhodococcus/enzimologia , Biocatálise , Escherichia coli/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Rhodococcus/genéticaRESUMO
Ene-reductases (ERs), or Old Yellow Enzymes, catalyze the asymmetric reduction of various activated alkenes. This class of biocatalysts is considered an attractive alternative to current chemical technologies for hydrogenation due to their high selectivity and specificity. Here the X-ray crystal structure of RmER, a "thermophilic"-like ER from Ralstonia (Cupriavidus) metallidurans, is reported. Unlike other members of this class of ERs, RmER is monomeric in solution which we previously related to its atypical elongated C-terminus. A typical dimer interface was however observed in our crystal structure, with the conserved Arg-"finger" forming part of the adjacent monomer's active site and the elongated C-terminus extending into the active site through contacting the "capping" domain. This dimerization also resulted in the loss of one FMN cofactor from each dimer pair. This potential transient dimerization and dissociation of FMN could conceivably explain the rapid rates previously observed when an FMN light-driven cofactor regeneration system was used during catalysis with RmER.
Assuntos
Proteínas de Bactérias/química , NADPH Desidrogenase/química , Ralstonia/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Modelos Moleculares , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Oxirredução , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Ralstonia/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
A deeper understanding of the >99 % S-selective reduction of both isomers of citral catalyzed by NCR ene reductase was achieved by active-site mutational studies and docking simulation. Though structurally similar, the E/Z isomers of citral showed a significantly varying selectivity response to introduced mutations. Although it was possible to invert (E)-citral reduction enantioselectivity to ee 46 % (R) by introducing mutation W66A, for (Z)-citral it remained ≥88 % (S) for all single-residue variants. Residueâ 66 seems to act as a lever for opposite binding modes. This was underlined by a W66A-based double-mutant library that enhanced the (E)-citral derived enantioselectivity to 63 % (R) and significantly lowered the S selectivity for (Z)-citral to 44 % (S). Formation of (R)-citronellal from an (E/Z)-citral mixture is a desire in industrial (-)-menthol synthesis. Our findings pave the way for a rational enzyme engineering solution.
Assuntos
Proteínas Fúngicas/química , Monoterpenos/química , Oxirredutases/química , Monoterpenos Acíclicos , Domínio Catalítico , Proteínas Fúngicas/genética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/genética , Mutação Puntual , Engenharia de Proteínas , Saccharomyces/enzimologia , Estereoisomerismo , Zymomonas/enzimologiaRESUMO
The use of pheromones in the integrated pest management of insects is currently considered a sustainable and environmentally benign alternative to hazardous insecticides. 4-Methylheptan-3-ol is an interesting example of an insect pheromone, because its stereoisomers are active towards different species. All four possible stereoisomers of this compound were prepared from 4-methylhept-4-en-3-one by a one-pot procedure in which the two stereogenic centres were created during two sequential reductions catalysed by an ene-reductase (ER) and an alcohol dehydrogenase (ADH), respectively.
Assuntos
Inseticidas/síntese química , Álcool Desidrogenase/metabolismo , Animais , Catálise , Insetos/efeitos dos fármacos , Inseticidas/química , Inseticidas/farmacologia , Feromônios/química , EstereoisomerismoRESUMO
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,ß-unsaturated compounds.