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Stereospecific carbonyl reductases

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Stereospecific carbonyl reductases

Stereospecific carbonyl reductases SCR1, SCR2, and SCR3 are described herein as are nucleotide sequences that encode these reductases. These stereospecific carbonyl reductases have anti-Prelog selectivity and have specificities that are useful for fine biochemical synthesis.

Inventors: Gaetano T. Montelione, Rong Xiao, Yao Nie, Yan Xu
USPTO Applicaton #: #20120270285 - Class: 435135 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Preparing Oxygen-containing Organic Compound >Carboxylic Acid Ester

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The Patent Description & Claims data below is from USPTO Patent Application 20120270285, Stereospecific carbonyl reductases.

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This patent application claims the benefit of priority of U.S. application Ser. No. 61/219,610, filed Jun. 23, 2009, which application is herein incorporated by reference.


This invention was made with government support under Grant #U54 GM074958 awarded by the National Institutes of General Medical Science, Protein Structure Initiative program. The government has certain rights in the invention.


The NAD(P)H-dependent carbonyl reductases catalyze reduction of a variety of endogenous and xenobiotic carbonyl compounds, including biologically and pharmacologically active substrates (Forrest et al., Chem. Biol. Interact., 129, 21-40 (2000)). There is considerable interest in the use of carbonyl reductases in the pharmaceutical and fine chemicals industries for the production of chiral alcohols, which are important building blocks for the synthesis of chirally-pure compounds, e.g., pharmaceutical agents (Panke et al., Curr. Opin. Biotechnol., 15, 272-279 (2004); Schmid et al., Nature, 409, 258-268 (2001); and Schoemaker et al., Science, 299, 1694-1697 (2003)). For such chiral auxiliaries, production from their corresponding prochiral ketones, the use of carbonyl reductases has advantages over chemo-catalysts in terms of their highly chemo-, enantio-, and regioselectivities. These features make stereospecific carbonyl reductases very useful from both scientific and industrial perspectives (Kroutil et al., Curr. Opin. Chem. Biol., 8, 120-126 (2004)). However, the range of current applications for stereospecific carbonyl reductases remains modest. This can be attributed to several limitations, including the stereospecificity and availability of enzymes. In addition, research on molecular mechanisms of oxidoreductases is still in its infancy. Further, most enzymes that can catalyze asymmetric reductions generally follow Prelog\'s rule in terms of stereochemical outcomes (Bradshaw et al., J. Org. Chem., 57, 1526-1532 (1992); Ernst et al., Appl. Microbiol. Biotechnol., 66, 629-634 (2005); Niefind et al., J. Mol. Biol., 327, 317-328 (2003); Prelog, Pure Appl. Chem., 9, 119-130 (1964)). Enzymes with anti-Prelog stereospecificity are quite rare, and only few have been isolated and characterized in purified forms (De Wildeman et al., Acc. Chem. Res. 40, 1260-1266, (2007)). Accordingly, stereospecific carbonyl reductases are needed. In particular, stereospecific carbonyl reductases with anti-Prelog stereospecificity are needed.



Accordingly, as described herein, three stereospecific carbonyl reductase genes (scr1, scr2, and scr3) from C. parapsilosis have been discovered. These genes have been cloned and expressed, and the encoded proteins purified to homogeneity and confirmed to function as stereospecific carbonyl reductases (SCR1, SCR2, and SCR3). These stereospecific carbonyl reductases have anti-Prelog selectivity and convert 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol (PED). These oxidoreductases have useful specificities that are useful for fine biochemical synthesis.

Application of biocatalysis in the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes. This is due to environmentally benign reaction conditions for biocatalysis and unparalleled chemo-, regio- and stereoselectivities. The newly identified stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol (PED) from 2-hydroxyacetophenone with NADPH as the coenzyme. The enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereo selectivity. Of the enzymes encoded in the gene cluster, SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities to ethyl 4-chloro-3-oxobutyrate, which affords ethyl 4-chloro-3-hydroxybutyrate, a precursor of the chiral side chain in the synthesis of atorvastatin (Lipitor®) and rosuvastatin, e.g., rosuvastatin calcium (Crestor®).


FIG. 1. Map of contig005802 of Candida parapsilosis genome including the four open reading frames, scr1, scr2, scr3, and cpadh.

FIG. 2. Amino acid sequence alignment of CPADH (GenBank accession number DQ675534; SEQ ID NO:1), SCR1 (GenBank accession number FJ939565; SEQ ID NO:4), SCR2 (GenBank accession number FJ939563; SEQ ID NO:3), and SCR3 (GenBank accession number FJ939564; SEQ ID NO:2) from C. parapsilosis. Gaps in the aligned sequences are indicated by dashes. Identical amino acid residues are enclosed in boxes. The conserved sequences of the cofactor-binding motif Gly-X-X-X-Gly-X-Gly (SEQ ID NO:9) and the catalytic tetrad of Asn-Ser-Tyr-Lys (SEQ ID NO:10) in the majority of SDRs are marked with arrows.

FIG. 3. Analysis of the overexpression of SCR1, SCR2, and SCR3. The proteins were separated on a 12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue G-250. Lane 1, total protein for SCR1; Lane 2, soluble fraction for SCR1; Lane 3, total protein for SCR2; Lane 4, soluble fraction for SCR2; Lane 5, total protein for SCR3; Lane 6, soluble fraction for SCR3; Lane 7, molecular mass standard.

FIG. 4. SDS-PAGE analysis of purified enzymes. The purified proteins were resolved by SDS-PAGE on a 12% polyacrylamide gel and stained with Coomassie Brilliant Blue G-250. Lane 1, molecular mass standard; Lane 2, purified SCR1; Lane 3, purified SCR2; Lane 4, purified SCR3.

FIG. 5. pH dependence of SCR1, SCR2, and SCR3 catalyzing 2-hydroxyacetophenone reduction. The enzyme activities of SCR1 (squares), SCR2 (triangles), and SCR3 (circles) were measured in 0.1 M acetate buffer (pH 4.0 to 6.0) or 0.1 M sodium phosphate buffer (pH 6.0 to 8.0) or 0.1 M Tris-HCl buffer (pH 8.0 to 8.5) with 2-hydroxyacetophenone as the substrate and NADPH as the cofactor. Maximal enzyme activity observed was set as 100% relative activity for each enzyme.

FIG. 6A-6E. Asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to 1-phenyl-1,2-ethanediol (PED) enantiomer by SCR1, SCR2, and SCR3, respectively. (6A) Standard sample of (R)-PED. (6B) Standard sample of (S)-PED. (6C) SCR1 catalyzed asymmetric reduction of 2-HAP. (6D) SCR2 catalyzed asymmetric reduction of 2-HAP. (6E) SCR3 catalyzed asymmetric reduction of 2-HAP.

FIG. 7A-7D. Substrate specificity of SCR1 and SCR3. The enzyme activities of SCR1 (open bars) and SCR3 (shaded bars) (7A) to various substrates (7B-7D) were measured as described herein. Maximal enzyme activity observed was set as 100% relative activity for the enzymes to various substrates.


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Application #
US 20120270285 A1
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Other USPTO Classes
435189, 536 232, 4353201, 43525233, 435157, 435156
International Class

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