| Production of tetraketide lactones by mutated Antirrhinum majus chalcone synthases (AmCHS1). | |
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MedLine Citation:
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PMID: 20547380 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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 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. |
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Authors:
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Masayoshi Hatayama; Hideaki Unno; Masami Kusunoki; Seiji Takahashi; Tokuzo Nishino; Toru Nakayama |
Publication Detail:
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Type: Journal Article Date: 2010-03-27 |
Journal Detail:
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Title: Journal of bioscience and bioengineering Volume: 110 ISSN: 1347-4421 ISO Abbreviation: J. Biosci. Bioeng. Publication Date: 2010 Aug |
Date Detail:
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Created Date: 2010-07-06 Completed Date: 2010-12-07 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 100888800 Medline TA: J Biosci Bioeng Country: Japan |
Other Details:
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Languages: eng Pagination: 158-64 Citation Subset: IM |
Copyright Information:
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Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved. |
Affiliation:
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Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai, Miyagi 980-8579 Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Acyltransferases
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chemistry*,
physiology* Antirrhinum / enzymology*, genetics* Cyclohexanones / chemical synthesis* Disaccharides / chemical synthesis* Enzyme Activation Enzyme Stability Lactones / chemical synthesis* Mutation / genetics Protein Engineering / methods* |
| Chemical | |
Reg. No./Substance:
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0/Cyclohexanones; 0/Disaccharides; 0/Lactones; 0/tetraketide; EC 2.3.-/Acyltransferases; EC 2.3.1.74/flavanone synthetase |
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