Purification and Characterization of 3-Oxoacyl-CoA Synthase of Mycobacterium smegmatis

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Abstract
3-Oxoacyl-CoA synthase, that condenses malonyl-CoA to other acyl-CoAs and takes part in the malonyl-CoA-dependent, acyl carrier protein (ACP)-non-requring fatty acid elongation system (“fatty acid elongation system II or elongation system II” (Kikuchi, S. & Kusaka, T. (1982) J. Biochem. 92,839–844)), was purified to homogeneity for the first time from the crude extract of Mycobacterium smegmatis by columnchromatographies. The molecular weight of this enzyme was estimated to be around 64,000 by Sephacryl S-300 gel filtration and 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic product from malonyl-CoA and stearoyl-CoA was identified as 3-oxoeicosanoyl-CoA by mass-spectrometry. Km values of the enzyme for malonyl-CoA and stearoyl-CoA were 41.7 μM and 52.6 μM, respectively. The enzyme was more active toward acyl-CoAs having acyl-carbon-numbers of 18 or more, either saturated or monounsaturated, than those with below 18. Cerulenin, a specific inhibitor of 3-oxoacyl-ACP synthase [EC 2.3.1.41], had no effect on this enzyme but iodoacetamide and N-ethylmaleimide (NEM) showed inhibitory effects.