DISSOCIATION OF DIMERIC 6-HYDROXYMELLEIN SYNTHASE, A POLYKETIDE BIOSYNTHETIC ENZYME IN CARROT CELL-EXTRACTS, WITH LOSS OF KETO-REDUCING ACTIVITY

6-Hydroxymellein synthase, an inducible polyketide biosynthetic enzyme in carrot cell extracts, is composed of two identical subunits, and t he homodimer is dissociated to monomeric peptides under high-ionic-str ength conditions with loss of the synthase activity. Appreciable radio activities were associated with the synthase proteins when the monomer enzyme was incubated with the radiolabeled substrates, acetyl-coenzym e A (CoA) and malonyl-CoA. Therefore, it appeared that the synthase do es not lose the ability of binding the substrate even after the dissoc iation to monomers. The monomeric synthase liberated triacetic acid la ctone as the derailment product instead of 6-hydroxymellein from the e nzyme-attached triketomethylene chain which is the immediate precursor of an NADPH-dependent keto-reducing reaction involved in 6-hydroxymel lein biosynthesis. These observations strongly suggest that the monome ric synthase retains the ability of ketomethylene chain elongation by the condensation of acyl-CoAs, but is lacking in an NADPH-dependent ke to-reducing activity toward the triketide intermediate. Results obtain ed in the present experiments imply that the catalytic domain of acyl- CoA condensation is able to associate with that of keto reduction, pos sibly belonging to another subunit, only in the homodimeric structure to organize the multicatalytic reaction center. (C) 1995 Academic Pres s, Inc.