Mechanism of Chalcone Synthase

Abstract

Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys164-His303) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys164 in a pH-dependent manner (pK a = 5.50). The acidic pK a of Cys164 suggests that an ionic interaction with His303 stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His303 maintains catalytic activity but shifts the pK a of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [14C]iodoacetamide into this mutant exhibits a pK a = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.