Incorporation of [2(14)C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells.

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. (14)C-Fatty acids synthesized by the extract from [2-(14)C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-(14)C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg(-1) protein·h(-1) occurred at a substrate level of 73 μM malonyl CoA, cofactor levels of 500 μM NADPH, 30 μg·ml(-1) E. coli ACP, and 1.0 mg·ml(-1) extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual (14)C-fatty acids were regulated by concentrations of [(14)C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [(14)C]palmitic acid were rapidly saturated at a low substrate level (0.3 μM malonyl CoA). Increasing the level of [2-(14)C]malonyl CoA permitted further synthesis of [(14)C]stearate and [(14)C]oleate. Desaturation of [(14)C]stearate to [(14)C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.