FreshPatents.com Logo
stats FreshPatents Stats
1 views for this patent on FreshPatents.com
2012: 1 views
Updated: August 12 2014
newTOP 200 Companies filing patents this week


    Free Services  

  • MONITOR KEYWORDS
  • Enter keywords & we'll notify you when a new patent matches your request (weekly update).

  • ORGANIZER
  • Save & organize patents so you can view them later.

  • RSS rss
  • Create custom RSS feeds. Track keywords without receiving email.

  • ARCHIVE
  • View the last few months of your Keyword emails.

  • COMPANY DIRECTORY
  • Patents sorted by company.

Follow us on Twitter
twitter icon@FreshPatents

Stereospecific carbonyl reductases

last patentdownload pdfdownload imgimage previewnext patent


20120270285 patent thumbnailZoom

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

view organizer monitor keywords


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

last patentpdficondownload pdfimage previewnext patent

CROSS-REFERENCE TO RELATED APPLICATION(S)

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.

STATEMENT OF GOVERNMENT SUPPORT

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.

BACKGROUND

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.

SUMMARY

OF CERTAIN EMBODIMENTS OF THE INVENTION

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®).

BRIEF DESCRIPTION OF THE FIGURES

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.

DETAILED DESCRIPTION

Certain embodiments of the present invention provide a purified polypeptide, the sequence of which comprises an amino acid sequence that has at least 70% identity to a Candida parapsilosis stereospecific carbonyl reductase, wherein the polypeptide has carbonyl reductase activity and does not comprise SEQ ID NO:1.

In certain embodiments, the amino acid sequence has at least 70% identity to at least one of the Candida parapsilosis stereospecific carbonyl reductases represented by SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In certain embodiments, the amino acid sequence has at least 70% identity to SEQ ID NO:2.

In certain embodiments, the amino acid sequence has at least 70% identity to SEQ ID NO:3.

In certain embodiments, the amino acid sequence has at least 70% identity to SEQ ID NO:4.

In certain embodiments, the amino acid sequence has at least 75% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence has at least 80% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence has at least 85% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence has at least 90% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence has at least 95% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence has at least 99% identity to the Candida parapsilosis stereospecific carbonyl reductase (e.g., to at least one of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4).

In certain embodiments, the amino acid sequence comprises SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

In certain embodiments, the amino acid sequence comprises SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

In certain embodiments, the amino acid sequence comprises SEQ ID NO:2.

In certain embodiments, the amino acid sequence comprises SEQ ID NO:3.

In certain embodiments, the amino acid sequence comprises SEQ ID NO:4.

In certain embodiments, the sequence of the polypeptide consists essentially of, or consists of, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In certain embodiments, the carbonyl reductase activity of the polypeptide is NADPH-dependent.

In certain embodiments, the polypeptide is an anti-Prelog-type stereospecific carbonyl reductase.

Certain embodiments of the present invention provide composition comprising the polypeptide as described herein.

Certain embodiments of the present invention provide an isolated nucleic acid sequence comprising a sequence that encodes a polypeptide described herein.

In certain embodiments, the sequence comprises SEQ ID NO:6 or of a degenerate variant of SEQ ID NO:6.

In certain embodiments, the sequence comprises SEQ ID NO:7 or of a degenerate variant of SEQ ID NO:7.

In certain embodiments, the sequence comprises SEQ ID NO:8 or of a degenerate variant of SEQ ID NO:8.

In certain embodiments, the sequence encodes SEQ ID NO:2.

In certain embodiments, the sequence encodes SEQ ID NO:3.

In certain embodiments, the sequence encodes SEQ ID NO:4.

Certain embodiments of the present invention provide an expression vector comprising an expression cassette operably linked to a nucleic acid molecule as described herein.

Certain embodiments of the present invention provide a host cell comprising a vector as described herein.

Certain embodiments of the present invention provide a method of reducing a carbonyl substrate, comprising contacting the substrate with a polypeptide described herein, or a composition described herein, in conditions suitable to catalyze the reduction of the carbonyl substrate. As used herein, a “carbonyl substrate” is a substrate that comprises at least one carbonyl group, such as a compound that comprises an α-ketoester, a β-ketoester, an aryl ketone or an aliphatic ketone (see, e.g., FIG. 7). The polypeptide having carbonyl reductase activity reduces a carbonyl group of the carbonyl substrate.



Download full PDF for full patent description/claims.

Advertise on FreshPatents.com - Rates & Info


You can also Monitor Keywords and Search for tracking patents relating to this Stereospecific carbonyl reductases patent application.
###
monitor keywords



Keyword Monitor How KEYWORD MONITOR works... a FREE service from FreshPatents
1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored.
3. Each week you receive an email with patent applications related to your keywords.  
Start now! - Receive info on patent apps like Stereospecific carbonyl reductases or other areas of interest.
###


Previous Patent Application:
Method and system for robotic algae harvest
Next Patent Application:
Hollow fiber membrane module for use in production of chemical substance, and process for production of chemical substance
Industry Class:
Chemistry: molecular biology and microbiology
Thank you for viewing the Stereospecific carbonyl reductases patent info.
- - - Apple patents, Boeing patents, Google patents, IBM patents, Jabil patents, Coca Cola patents, Motorola patents

Results in 0.57844 seconds


Other interesting Freshpatents.com categories:
Amazon , Microsoft , IBM , Boeing Facebook

###

Data source: patent applications published in the public domain by the United States Patent and Trademark Office (USPTO). Information published here is for research/educational purposes only. FreshPatents is not affiliated with the USPTO, assignee companies, inventors, law firms or other assignees. Patent applications, documents and images may contain trademarks of the respective companies/authors. FreshPatents is not responsible for the accuracy, validity or otherwise contents of these public document patent application filings. When possible a complete PDF is provided, however, in some cases the presented document/images is an abstract or sampling of the full patent application for display purposes. FreshPatents.com Terms/Support
-g2-0.2018
     SHARE
  
           

FreshNews promo


stats Patent Info
Application #
US 20120270285 A1
Publish Date
10/25/2012
Document #
13379942
File Date
06/23/2010
USPTO Class
435135
Other USPTO Classes
435189, 536 232, 4353201, 43525233, 435157, 435156
International Class
/
Drawings
7



Follow us on Twitter
twitter icon@FreshPatents