Structure-Guided Programming of Polyketide Chain-Length Determination in Chalcone Synthase

Abstract

Chalcone synthase (CHS) belongs to the family of type III polyketide synthases (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 malonyl-CoA. Thus, position 256 serves as an ideal target to probe the link between cavity volume and polyketide chain-length determination in type III PKS. Functional examination of CHS G256A, G256V, G256L, and G256F mutants reveals altered product profiles from that of wild-type CHS. With p-coumaroyl-CoA as a starter molecule, the G256A and G256V mutants produce notably more tetraketide lactone. Further restrictions in cavity volume such as that seen in the G256L and G256F mutants yield increasing levels of the styrylpyrone bis-noryangonin from a triketide intermediate. X-ray crystallographic structures of the CHS G256A, G256V, G256L, and G256F mutants establish that these substitutions reduce the size of the active site cavity without significant alterations in the conformations of the polypeptide backbones. The side chain volume of position 256 influences both the number of condensation reactions during polyketide chain extension and the conformation of the triketide and tetraketide intermediates during the cyclization reaction. These results viewed in conjunction with the natural sequence variation of residue 256 suggest that rapid diversification of product specificity without concomitant loss of substantial catalytic activity in related CHS-like enzymes can occur by site-specific evolution of side chain volume at position 256.

Crystal Structure of a Bacterial Type III Polyketide Synthase and Enzymatic Control of Reactive Polyketide Intermediates

“…Although mutation to Phe or Trp was tolerated by THNS, nonconservative mutations at this position abrogated the enzymatic loading of the physiological malonyl starter. These results are not surprising, since the substitution of bulky side chains in place of the spatially equivalent Gly 256 residue of alfalfa CHS was shown to affect both starter specificity and the number of catalyzed polyketide chain extension steps (22,36). As first observed in the crystal structure of 2-PS (22), which loads an acetyl moiety as a starter, variation of FIG.…”

Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity

“…Use of 4-methylcinnamoyl-CoA produces a triketide styrylpyrone. With feruloyl-CoA, benzoylCoA, phenylacetyl-CoA, and aliphatic CoA-thioesters, CHS generates tetraketide and triketide lactones (7,17). The k cat ͞K m values for different starter molecules confirm that alfalfa CHS2 prefers cinnamoyl-derived CoAs and aliphatic CoAs of 6-8 carbons (Table 2).…”

“…This observation indicates that the conformation of the tetraketide intermediate differs from that found in either the CHS or ACS active site. Recent experiments demonstrate that mutations of residues lining the active site cavity or use of unnatural starter molecules result in production of polyketide lactones by type III PKS (7,(16)(17)(18)30). Because the overall three-dimensional folds of CHS and the F215S mutant are identical, the structural differences between the N-methylanthraniloyl-and the pcoumaroyl-moieties of the tetraketide likely alter how the starter molecule fits within the active site.…”

“…Unlike the larger 6-deoxyerythronolide and actinorhodin PKS of bacterial origin (3), the type III PKS are homodimeric enzymes that orchestrate a series of acyltransferase, decarboxylation, condensation, cyclization, and aromatization reactions at two functionally independent active sites (4). Crystallographic and protein engineering studies on Medicago sativa (alfalfa) CHS2 and Gerbera hybrida 2-pyrone synthase demonstrate that the structure of the type III PKS active site dictates starter molecule preference, polyketide chainlength, and regio-specificity of polyketide cyclization (5)(6)(7). This architecturally simple molecular organization forms an attractive three-dimensional scaffold both for understanding the structural basis for rapid diversification of biosynthetic activities in plants and for guiding structure-assisted engineering experiments aimed at altering the biosynthetic activities of these PKS (8,9).…”

Site-directed Mutagenesis of Benzalacetone Synthase

“…2) (11)(12)(13)(14)(15)(16)(17). The catalytic center of CHS is composed of four amino acid residues: the catalytic triad of Cys-164, His-303, and Asn-336 and the "gatekeeper" Phe-215, absolutely conserved in all of the known type III PKSs.…”