Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus

Abstract

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces coUinus. This enzyme, a dimer with subunits of identical Mr (36,000), exhibits a Km of 1.5 0.3 ,uM for NADPH and 25 + 3 ,uM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30°C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained.

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus

“…The recombinant ChcA was purified fourfold from the E. coli BL21(pLysS)/pPW5-induced culture. The final specific activity of the purified protein was 3667 mU/mg of protein, comparable to the specific activity of 3,400 mU/mg of protein for the protein originally purified from S. collinus (51). SDS-PAGE of the purified protein resulted in a single major band corresponding to a molecular mass of 37 Ϯ 2 kDa (Fig.…”

“…No measurable activity could be obtained with cell extracts obtained from fermentations of E. coli BL21(pLysS) not carrying pPW5. Cell extracts of S. collinus have previously been shown to contain a specific activity of ChcA of only 0.55 mU/mg of protein (51). Purification and analysis of the S. collinus ChcA from E. coli.…”

“…This ORF would encode a polypeptide of 280 amino acids and a predicted molecular mass of 29.7 kDa. A molecular mass of 36.0 kDa has previously been obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the ChcA enzyme isolated from S. collinus (51). The deduced amino acid sequence of this ORF includes all three previously determined internal tryptic peptides obtained for ChcA (Fig.…”

“…It is thought that most of the steps in this process occur with the carboxylic acid activated as a coenzyme A thioester and that the final step in the pathway is the conversion of 1-cyclohexenylcarbonyl coenzyme A (CoA) (compound 3) to cyclohexylcarbonyl CoA (compound 4) (42,43,(48)(49)(50). An enzyme, with the designation 1-cyclohexenylcarbonyl CoA reductase (ChcA), capable of catalyzing this ␣,␤-double bond reduction has been purified and characterized from S. collinus (51).…”

“…ChcA was assayed by spectrophotometric determination of NADPH oxidation at 340 nm in buffer C (consisting of 50 mM potassium phosphate and 10% [wt/vol] glycerol at pH 7.3) as previously described (50,51). Coenzyme A thioesters were prepared by using ethylchlorofor- mate as previously described (50,51).…”