In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Abstract

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1- 14C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C 32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1- 14C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C 34:0 + C 34:1) mycolic acids and the other to (C 36:0 + C 36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C 32:0 mycolic acid synthesized by [1- 14C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1- 14C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from 14C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1- 14] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.