title: Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1).
abstract: Chalcone synthase (CHS) is a key enzyme of flavonoid biosynthesis in higher plants, catalyzing the stepwise decarboxylative condensation of three acetate units from malonyl-CoA with p-coumaroyl-CoA to yield 2',4,4',6'-tetrahydroxychalcone (THC). Reaction (at pH 7.5) of a mutant (V196M/T197A) of Antirrhinum majus CHS (AmCHS1) with p-coumaroyl-CoA and malonyl-CoA yielded a significant amount of a non-chalcone product, along with a small amount of THC. The non-chalcone product was identified as p-coumaroyltriacetic acid lactone (CTAL), a tetraketide lactone produced due to derailment from the canonical THC-producing reaction pathway. In vitro, the wild-type AmCHS1 showed low CTAL-producing activity at pH 7.5, but an appreciable level at pH 10. Each of the amino acid substitutions, V196M, T197A and V196M/T197A, caused a shift toward neutrality of the optimum pH for CTAL-producing activity. The V196M substitution resulted in a loss of THC-producing activity, as well as a 12.6-fold enhancement of CTAL-producing activity (at pH 7.5); hence, AmCHS1 was converted to a p-coumaroyltriacetic acid synthase by this single amino acid substitution. The THC-producing activity of the V196M mutant appeared to be restored by additional T197A substitution, although a single T197A substitution caused no substantial enhancement of the CTAL-producing activity of the wild-type enzyme. The enhancement of the tetraketide producing activity upon V196M and V196M/T197A substitutions was most markedly observed when p-coumaroyl-CoA was used as the starter substrate, and only slightly with benzoyl-, caffeoyl- and hexanoyl-CoAs. These results show the importance of the two contiguous amino acids at positions 196 and 197 for product specificity of an AmCHS1-catalyzed reaction.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai, Miyagi 980-8579 Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Hatayama
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.forename: Masami
authors.initials: M
authors.lastname: Kusunoki
authors.forename: Seiji
authors.initials: S
authors.lastname: Takahashi
authors.forename: Tokuzo
authors.initials: T
authors.lastname: Nishino
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000502:physiology*; D032161:Antirrhinum / Q000201:enzymology* / Q000235:genetics*; D003512:Cyclohexanones / Q000138:chemical synthesis*; D004187:Disaccharides / Q000138:chemical synthesis*; D004789:Enzyme Activation; D004795:Enzyme Stability; D007783:Lactones / Q000138:chemical synthesis*; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: D003512:Cyclohexanones; D004187:Disaccharides; D007783:Lactones; C413265:tetraketide; D000217:Acyltransferases; C020141:flavanone synthetase