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J. Biol. Chem., Vol. 275, Issue 50, 39640-39646, December 15, 2000

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Mechanism of Chalcone Synthase
pK_a OF THE CATALYTIC CYSTEINE AND THE ROLE OF THE CONSERVED HISTIDINE IN A PLANT POLYKETIDE SYNTHASE^*

Joseph M. Jez and Joseph P. Noel^§

From the Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

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.

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