Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells

6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation acid the ketoreduction in the presence of the nucleotide reducing co-factor s. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable in hibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of th e NADH-associated enzyme while, in the presence of NADPH, the compound slig htly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deute rium-labeled compound, however, the kH/kD value was markedly reduced if NAD H and [D]NADH were employed as the reducing co-factors. These results sugge st that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an i mportant role in the regulation of the enzyme activity. (C) 2000 Elsevier S cience Ireland Ltd. All rights reserved.