Purification and characterization of 3-oxoacyl-CoA synthase of Mycobacterium smegmatis.
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-requiring 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 column-chromatographies. 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 microM and 52.6 microM, 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.
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000302:isolation & purification* / Q000378:metabolism; D000217:Acyltransferases / Q000302:isolation & purification*; D002569:Cerulenin / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D009161:Mycobacterium / Q000201:enzymology*; D013379:Substrate Specificity
D002569:Cerulenin; D000217:Acyltransferases; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D005033:Ethylmaleimide; D007460:Iodoacetamide