Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.     

Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway

Alkylresorcinol moieties of cannabinoids are derived from olivetolic acid (OLA), a polyketide metabolite. However, the polyketide synthase (PKS) responsible for OLA biosynthesis has not been identified. In the present study, a cDNA encoding a novel PKS, olivetol synthase (OLS), was cloned from Cannabis sativa. Recombinant OLS did not produce OLA, but synthesized olivetol, the decarboxylated form of OLA, as the major reaction product. Interestingly, it was also confirmed that the crude enzyme extracts from flowers and rapidly expanding leaves, the cannabinoid-producing tissues of C. sativa, also exhibited olivetol-producing activity, suggesting that the native OLS is functionally expressed in these tissues. The possibility that OLS could be involved in OLA biosynthesis was discussed based on its catalytic properties and expression profile.