Structure-guided programming of polyketide chain-length determination in chalcone synthase

Chalcone synthase (CHS) belongs to the family of type III polyketide syntha ses (PKS) that catalyze formation of structurally diverse polyketides. CHS synthesizes a tetraketide by sequential condensation of three acetyl anions derived from malonyl-CoA decarboxylation to a p-coumaroyl moiety attached to an active site cysteine. Gly256 resides on the surface of the CHS active site that is in direct contact with the polyketide chain derived from malo nyl-CoA. Thus, position 256 serves as an ideal. target to probe the link be tween cavity volume and polyketide chain-length determination in type III P KS. Functional examination of CHS G256A, G256V, G256L, and G256F mutants re veals altered product profiles from that of wild-type CHS. With p-coumaroyl -CoA as a starter molecule, the G256A and G256V mutants produce notably mor e tetraketide lactone. Further restrictions in cavity volume such as that s een in the G256L and G256F mutants yield increasing levels of the styrylpyr one bis-noryangonin from a triketide intermediate. X-ray crystallographic s tructures of the CHS G256A, G256V, G256L, and G256F mutants establish that these substitutions reduce the size of the active site cavity without signi ficant alterations in the conformations of the polypeptide backbones. The s ide chain volume of position 256 influences both the number of condensation reactions during polyketide chain extension and the conformation of the tr iketide and tetraketide intermediates during the cyclization reaction. Thes e results viewed in conjunction with the natural sequence variation of resi due 256 suggest that rapid diversification of product specificity without c oncomitant loss of substantial catalytic activity in related CHS-like enzym es can occur by site-specific evolution of side chain volume at position 25 6.