Identification, cloning, and characterization of a novel ketoreductase from the cyanobacterium Synechococcus sp. strain PCC 7942.
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]).
Hölsch
Kathrin
K
Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Garching, Germany.
Havel
Jan
J
Haslbeck
Martin
M
Weuster-Botz
Dirk
D
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
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009249:NADP
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