title: Identification, cloning, and characterization of a novel ketoreductase from the cyanobacterium Synechococcus sp. strain PCC 7942.
abstract: A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44 degrees C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 +/- 2.15 U mg(-1)) and 2',3',4',5',6'-pentafluoroacetophenone (8.57 +/- 0.49 U mg(-1)) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2',3',4',5',6'-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).
authors.affiliation: Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Garching, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Hölsch
authors.forename: Jan
authors.initials: J
authors.lastname: Havel
authors.forename: Martin
authors.initials: M
authors.lastname: Haslbeck
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D052196:Levilactobacillus brevis / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D046940:Synechococcus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009249:NADP
references.citation: Appl Microbiol Biotechnol. 2007 Jul;75(5):1031-7
references.pmid: 17345080
references.citation: J Biol Chem. 1992 Mar 25;267(9):5751-4
references.pmid: 1556094
references.citation: Chem Commun (Camb). 2002 Aug 21;(16):1782-3
references.pmid: 12196997
references.citation: J Biochem Biophys Methods. 2008 Apr 24;70(6):850-6
references.pmid: 18201766
references.citation: Protein Eng. 2001 Dec;14(12):993-1000
references.pmid: 11809930
references.citation: Anal Biochem. 1994 Mar;217(2):220-30
references.pmid: 8203750
references.citation: Electrophoresis. 2001 Aug;22(14):2955-68
references.pmid: 11565790
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: J Med Chem. 1997 Apr 25;40(9):1347-65
references.pmid: 9135032
references.citation: Appl Microbiol Biotechnol. 2005 Mar;66(6):629-34
references.pmid: 15549291
references.citation: Biotechnol Bioeng. 2006 Dec 20;95(6):1177-87
references.pmid: 16933330
references.citation: Biochemistry. 2001 Oct 30;40(43):12772-81
references.pmid: 11669613
references.citation: Structure. 2000 Apr 15;8(4):339-47
references.pmid: 10801480
references.citation: Microbiology (Reading). 2002 Apr;148(Pt 4):951-960
references.pmid: 11932442
references.citation: Appl Microbiol Biotechnol. 2003 Apr;61(2):133-9
references.pmid: 12655455
references.citation: Biochem J. 2006 Jan 15;393(Pt 2):447-57
references.pmid: 16225460
references.citation: Curr Opin Microbiol. 2005 Apr;8(2):149-53
references.pmid: 15802245
references.citation: Electrophoresis. 1988 Jan;9(1):28-32
references.pmid: 2466645
references.citation: J Phycol. 1968 Mar;4(1):1-4
references.pmid: 27067764
references.citation: Nature. 2001 Jan 11;409(6817):258-68
references.pmid: 11196655