Mechanism of chalcone synthase. pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase.
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 (Cys(164)-His(303)) 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 Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) 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 [(14)C]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.
Jez
J M
JM
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Noel
J P
JP
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism*; D000477:Alkylating Agents / Q000494:pharmacology; D000838:Anions; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D013997:Time Factors
D000477:Alkylating Agents; D000838:Anions; D004791:Enzyme Inhibitors; D005973:Glutamine; D006639:Histidine; D000217:Acyltransferases; C020141:flavanone synthetase; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase; D003545:Cysteine; D007460:Iodoacetamide