Incorporation of [2C]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. C-Fatty acids synthesized by the extract from [2-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-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 protein·h occurred at a substrate level of 73 μM malonyl CoA, cofactor levels of 500 μM NADPH, 30 μg·ml E. coli ACP, and 1.0 mg·ml extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual C-fatty acids were regulated by concentrations of [C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [C]palmitic acid were rapidly saturated at a low substrate level (0.3 μM malonyl CoA). Increasing the level of [2-C]malonyl CoA permitted further synthesis of [C]stearate and [C]oleate. Desaturation of [C]stearate to [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. [ABSTRACT FROM AUTHOR]