Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1)(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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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 pH7.5) 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 pH7.5, but an appreciable level at pH10. Each of the amino acid substitutions, V196M, T197A and V196M/T197A, caused a shift toward neutrality of the optimum pH for CFAL-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 pH7.5); 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.
著者
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Hatayama Masayoshi
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Unno Hideaki
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University
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Kusunoki Masami
Department of Biotechnology, Faculty of Engineering, Yamanashi University
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Takahashi Seiji
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Nishino Tokuzo
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Nakayama Toru
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University
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Nishino Tokuzo
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Nishino T
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Nishino Tokuzo
Dep. Of Biochemistry And Engineering Graduate School Of Engineering Tohoku Univ. Aoba-yama Sendai Mi
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Nakayama Toru
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Takahashi Seiji
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Unno Hideaki
Department Of Applied Chemistry Faculty Of Engineering Nagasaki University
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Kusunoki Masami
Department Of Biotechnology Faculty Of Engineering Yamanashi University
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Hatayama Masayoshi
Department Of Biomolecular Engineering Graduate School Of Engineering Tohoku University
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Takahashi Seiji
Department Of Applied Chemistry Faculty Of Engineering Kyushu Institute Of Technology
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Nishino Tokuzo
Department Of Biochemistry And Engineering Faculty Of Engineering Tohoku University
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NAKAYAMA Toru
Department of Applied Microbial Technology, The Kumamoto Institute of Technology:(Present office)Institute for Fundamental Research, Suntory Ltd.
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