| Project/Area Number |
05044095
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Kyoto University |
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Principal Investigator |
SODA Kenji Institute for Chemical Research, Kyoto University, 化学研究所, 教授 (30027023)
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| Co-Investigator(Kenkyū-buntansha) |
RINGE Dagmer Brandeis University, 教授
MANNING James The Rockfeller University, 理学部, 教授
CHOU Hong-yon College of Natural Science, Korea University, 助教授
成 文喜 韓国科学技術研究所, 高等研究員
KURIHARA Tatsuo Institute for Chemical Research, Kyoto University, 化学研究所, 助手 (70243087)
YOSHIMURA Tohru Institute for Chemical Research, Kyoto University, 化学研究所, 助手 (70182821)
ESAKI Nobuyoshi Institute for Chemical Research, Kyoto University, 化学研究所, 助教授 (50135597)
SUNG Moon-hee Korean Institute of Science and Technology
DAGMER Ringe ブランダイズ大学, 教授
JAMES Mannin ロックフェラー大学, 理学部, 教授
谷沢 克行 大阪大学, 産業科学研究所, 助教授 (20133134)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1994: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1993: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | thermophilic bacteria / thermostable enzymes / D-amino acid transaminase / pyridoxal phosphate / X-ray crystal structure analysis / pyridoxal enzymes / screening / protein engineering |
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| Research Abstract |
We found that D-amino acid aminotransferase (D-AAT) of Bacillus sp.YM-1 and branched-chain L-amino acid aminotransferase (BCAT) of E.coli catalyze the re-face hydrogen transfer. These enzymes show a significant sequence homology with each other, but does not with all other aminotransferases. The three-dimensional structure of D-AAT demonstrated that the topographical situation of the catalytic residue of D-AAT is opposite to that of AspAT.The result of the crystallography also showed that the overall fold of D-AAT is quite different from those of AspAT and other aminotransferases, whose structures resemble each other. We established a simple method for determination of the stereospecificity of C-4' hydrogen transfer of the coenzyme based on these findings. We developed also a general procedure to determine the common free D-amino acids except D-proline by means of D-AAT and 2-oxohexanoate : the formation of norleucine denotes the presence of some D-amino acid (s) whose identity can be established by a corresponding decrease in the susceptible amino acid (s) after treatment. We found that D-AAT is inactivated by incubation with D-aspartate, D-glutamate and D-alanine, the best substrates, and that the inactivation is accompanied by the slow release of the cx-carboxylg group of these amino acids. Lys-145, which binds pyridoxal-P,is not involved in the inactivation since K145Q and K145N mutanat enzymes are also inactivated. We studied the catalytic role of Leu-201, the residue in the vicinity of the active site to interact with the bound pyridoxal-P.The L201A and L201W mutant enzymes showed anomalous kinetic behavior. These show that Leu-201 regulates the function of cofactor during the reaction of D-AAT.
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