Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli.
A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.
Wallace
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Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore MD, USA.
Bao
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Dai
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Digate
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Schuler
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Speedie
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Reynolds
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D044944:Acyl-CoA Dehydrogenases; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*
D009249:NADP; D010088:Oxidoreductases; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+)