Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones

The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.     

New Generation of Biocatalysts for Organic Synthesis

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts—reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases—will be discussed herein and exemplified by the syntheses of interesting compounds.     

Dithiolene complexes and the nature of molybdopterin

The development of the coordination chemistry of dithiolene ligands is summarised, together with a consideration of the electronic structure of complexes of these ‘non-innocent’ ligands. This information provides a context for a consideration of the role of dithiolenes in natural systems, i.e. as the ligand that binds molybdenum (or tungsten) at the catalytic centre of an extensive series of enzymes. These enzymes catalyse the transfer of an oxygen atom to or from the substrate: e.g. the sulfite oxidases catalyse the conversion of sulfite to sulfate and the nitrate reductases catalyse the conversion of nitrate to nitrite. The nature of the catalytic centres of several of these enzymes has been determined and each involves one or two ‘molybdopterin’ (MPT) cofactors bound to a mononuclear metal centre via their dithiolene group. The biosynthesis of MPT is described and, given its nature, possible roles for this moiety in the function of the oxotransferase enzymes are discussed. The review concludes with a consideration of the coordination chemistry that has been stimulated by the present knowledge of the nature and function of the catalytic centres of these enzymes.     

Enantioselective Enzymes by Computational Design and In Silico Screening

Computational enzyme design holds great promise for providing new biocatalysts for synthetic chemistry. A strategy to design small mutant libraries of complementary enantioselective epoxide hydrolase variants for the production of highly enantioenriched (S,S)‐diols and (R,R)‐diols is developed. Key features of this strategy (CASCO, catalytic selectivity by computational design) are the design of mutations that favor binding of the substrate in a predefined orientation, the introduction of steric hindrance to prevent unwanted substrate binding modes, and ranking of designs by high‐throughput molecular dynamics simulations. Using this strategy we obtained highly stereoselective mutants of limonene epoxide hydrolase after experimental screening of only 37 variants. The results indicate that computational methods can replace a substantial amount of laboratory work when developing enantioselective enzymes.     

Enzyme-catalysed nitrate reduction-themes and variations as revealed by protein film voltammetry

Protein film voltammetry has been used to define the catalytic performance of two nitrate reductases: the respiratory nitrate reductase, NarGH, from Paracoccus pantotrophus and the assimilatory nitrate reductase, NarB, from Synechococcus sp. PCC 7942. NarGH and NarB present distinct "fingerprints" of catalytic activity when viewed in this way. Potentials that provide insufficient driving force for significant rates of nitrate reduction by NarB result in appreciable rates of nitrate reduction by NarGH. However, both enzymes display complex modulations in their rate of substrate reduction when viewed across the electrochemical potential domain.     

Combinatorial libraries of biocatalysts: application and screening

Enzymes can perform intricate regioselective and/or enantioselective chemical transformations and can accelerate reaction rates by enormous factors all under mild conditions. However, enzymes almost always present problems for use on an industrial scale. Evolutionary design approaches can be applied to the generation of stable enzymes with improved or novel catalytic activities. Directed evolution can be considered as the biotechnological equivalent of combinatorial chemistry, where the expressed proteins are the combinatorial libraries of biocatalysts. This review will focus on the search of novel biocatalysts produced by genetic engineering with modified activity and stability in different environments, substrate specificity and enantioselectivity. Methods of screening and/or selection for the desired properties will also be described. Finally, the efforts in de novo enzyme design are mentioned.     

Optimization of hydrolase efficiency in organic solvents

The application of hydrolases in organic solvents for synthetic purposes is a procedure routinely adopted in organic chemistry, especially for the preparation of chiral building blocks. Numerous studies have shed light on several aspects of the mechanism of hydrolase action in low-water environments. Procedures suitable to improve the catalytic efficiency of enzymes and productivity of the synthetic processes have been reported. These fundamental and applied investigations have made hydrolase-catalyzed reactions in organic solvents of industrial interest. In this article we describe and discuss various approaches adopted to optimize the performance of hydrolases in organic media, with special emphasis on the formulation of the biocatalysts which, under proper conditions, can display an activity equal to that displayed in aqueous buffers.     

含光学活性联二萘酚基团可溶性高分子手性配体的合成及其不对称加成反应

不对称催化是有机化学研究的前沿领域和发展方向．近10年来,手性配体的研究得到了长足的发展,大量的小分子手性配体被报道,其中许多手性配体展示了优异的催化性能．但是由于昂贵的手性配体（金属催化剂）分离、回收和再利用的问题,从而限制了小分子催化剂的应用．因此,解决手性金属催化剂的分离与回收问题是不对称催化研究领域的一个热点问题．为了解决这一关键问题,国内外科学家从负载的角度出发已经成功地发展了众多的研究方法,无机负载法、交联高分子负载法、可溶性高分子负载法、树状大分子负载法等．许多负载的手性金属催化剂已经表现出优异的催化活性和高的对映选择活性．     

Systematic investigation of Saccharomyces cerevisiae enzymes catalyzing carbonyl reductions

Eighteen key reductases from baker's yeast (Saccharomyces cerevisiae) have been overproduced in Escherichia coli as glutathione S-transferase fusion proteins. A representative set of alpha- and beta-keto esters was tested as substrates (11 total) for each purified fusion protein. The stereoselectivities of beta-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both L- and D-alcohols with high stereoselectivities. While alpha-keto esters were generally reduced with lower enantioselectivities, it was possible in all but one case to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. Taken together, the results demonstrate not only that individual yeast reductases can be used to supply important chiral building blocks, but that GST-fusion proteins allow rapid identification of synthetically useful biocatalysts (along with their corresponding genes).     

Chiral discrimination by hydrolytic enzymes in the synthesis of optically pure materials

Enantioselection effected by commercially available hydrolytic enzymes reported in recent years from our own laboratory and those published by other groups is described. The discussion is confined to enantioselection in symmetric diols, amino acids and benzopyran derivatives only. The paper describes a variety of substrates accepted by these hydrolytic enzymes to produce compounds in high enantiomeric excess which can be used as chiral building blocks for the synthesis of compounds of pharmaceutical and synthetic interest.     

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).     

Organocatalytic Enantioselective Vinylogous Pinacol Rearrangement Enabled by Chiral Ion Pairing

An enantioselective pinacol rearrangement of functionalized (E)‐2‐butene‐1,4‐diols was developed. In the presence of a catalytic amount of a chiral BINOL‐derived N‐triflyl phosphoramide, these 1,4‐diols rearranged to β,γ‐unsaturated ketones in excellent yields and enantioselectivities. The formation of a chiral ion pair between the intermediary allylic cation and the chiral phosphoramide anion was postulated to be responsible for the highly efficient chirality transfer. These chiral building blocks were further converted into enantioenriched polysubstituted tetrahydrofuran and tetrahydronaphthalene derivatives.     

Emulation of racemase activity by employing a pair of stereocomplementary biocatalysts

Racemization is the key step to turn a kinetic resolution process into dynamic resolution. A general strategy for racemization under mild reaction conditions by employing stereoselective biocatalysts is presented, in which racemization is achieved by employing a pair of stereocomplementary biocatalysts that reversibly interconvert an sp3 to a sp2 center. The formal interconversion of the enantiomers proceeds via a prochiral sp2 intermediate the formation of which is catalyzed either by two stereocomplementary enzymes or by a single enzyme with low stereoselectivity. By choosing appropriate reaction conditions, the amount of the prochiral intermediate is kept to a minimum. This general strategy, which is applicable to redox enzymes (e.g., by acting on R2CHOH and R2CHNHR groups) and lyase-catalyzed addition-elimination reactions, was proven for the racemization of secondary alcohols by employing alcohol dehydrogenases. Thus, enantiopure chiral alcohols were used as model substrates and were racemized either with highly stereoselective biocatalysts or by using (rarely found) non-selective enzymes.     

Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

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.     

Aminoacyl-tRNA Synthetases: The Division into Two Classes Predicted by the Chemistry of Substrates and Enzymes

In biological systems, almost all chemical reactions are catalyzed by enzymes. In order to understand the mode of action of these biocatalysts, we need to know precise details of their structures, their active sites, and their functional groups. Most important are those parts of the protein molecule that are responsible for precise recognition of the substrates, for the specific interaction between the enzymes and their reactants. Decades can pass between the isolation of an enzyme and the determination of its exact structure by X-ray analysis. For the chemist, however, means are known by which initial information may be gathered relatively quickly: the synthesis of modified substrates and affinity labeling. During the last twenty five years these two methods have been put to the test on aminoacyl-tRNA synthetases. Today, we know enough about the structures of these enzymes to evaluate the success of this “chemistry on macromolecules,” or their substrates. Chemists have had some successes, which provided the structural analysts with valuable preliminary results.     

生物催化合成光学活性环氧化物

手性环氧化物及其相应的邻二醇在合成上具有重要应用价值。近年来,制取这些手性合成块的合成方法得到了很的大发展。本文着重讨论当前国际上采用生物催化合成光学活性环氧化物的发展状况。     

Stereoselective reduction of 2-butenolides to chiral butanolides by reductases from cultured cells of Glycine max

The stereoselective reduction of 2-butenolides by two reductases, p51 and p83, from cultured plant cells of Glycine max was investigated. The reduction of 2-methyl-2-butenolide by p51 reductase produced (R)-2-methylbutanolide, whereas the reduction by p83 reductase gave (S)-2-methylbutanolide. Both reductases reduced 3-methyl-2-butenolide to (R)-3-methylbutanolide. The reduction of 2,3-dimethyl-2-butenolide by p51 reductase gave (2R,3R)-2,3-dimethylbutanolide, whereas the reduction by p83 reductase produced (2S,3R)-2,3-dimethylbutanolide. The reduction of 4-alkyl-2-butenolides with these reductases was accompanied by resolution of chiral centers affording (R)-4-alkylbutanolides.     

Engineering and assaying of cytochrome P450 biocatalysts

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.     

Modern trends in biocatalytic synthesis of chiral compounds

Characteristic features and modern trends in the biocatalytic synthesis of chiral compounds have been discussed. New processes of biocatalytic synthesis use at least two enzymes. Whole cells of recombinant strains are utilized as biocatalysts, and the complete set of target enzymes can be expressed in a single cell.     

The direct amino acid-catalyzed asymmetric incorporation of molecular oxygen to organic compounds

We have disclosed the direct catalytic incorporation of 1O2 to aldehydes. The unprecedented amino acid-catalyzed asymmetric alpha-oxidation of aldehydes with molecular oxygen or air proceeded with high chemoselectivity and was a direct entry for the synthesis of both enantiomers of terminal diols. The results demonstrated that simple amino acids accomplished catalytic asymmetric oxidations with molecular oxygen or air, which has previously been considered to be in the domain of enzymes and chiral transition-metal complexes. The efficiency of the catalytic process may warrant the existence of an ancient pathway for the synthesis of hydroxylated organic compounds.