Crosslinking of two sulfhydryl groups mediating substrate channeling at the reaction center of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme

摘要

Activity of 6-hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme, was markedly inhibited by the treatment with 1,3-dibromopropan-2-one (DBP), a bifunctional crosslinking reagent. The substrate binding ability of DBP-modified 6HM synthase was quite similar to that of the enzyme treated with iodoacetoamide plus chloroacetyl-CoA (ClAcCoA) strongly suggesting that both Cys-SH of ketoacyl synthase and 4'-phosphopantetheine-SH attached to acyl carrier protein (ACP) were alkylated with this crosslinker. The molecular weight of the DBP-treated 6HM synthase as analyzed by SDS-PAGE was essentially identical to that of the monomer subunit of the synthase, and the chemical stoichiometry of the enzyme protein versus DBP in the synthase-crosslinker complex was estimated to be approximately 1: 1. These results suggest that the cysteamine-SH of ACP is able to move to within 5 Angstrom of the active Cys-SH to mediate the substrate channeling at the reaction center of this multifunctional enzyme. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved. [References: 15]