| Project/Area Number |
03670132
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
KURAMITSU Seiki Osaka Univ., Biology, Professor, 理学部, 教授 (60153368)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Enzyme / Catalytic Mechanism / Substrate Recogition / Protein Engineering / Aminotransferase / Chimera / X-Ray Crystallography / Site-Directed Mutagenesis / 量産化 / アスパラギン酸アミノトランスフェラ-ゼ |
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| Research Abstract |
Aminotransferases catalyze transamination between amino acids and alphaketo acids. Escherichia coli aspartate aminotransferase (AspAT) has high specificity for acidic substrates, whereas E. coli aromatic amino acid aminotransferase (AroAT) has high specificity for both acidic and hydrophobic substrates. The two proteins have only 40% amino acid sequence homology, and 50% nucleotide sequence homology. X-ray crystallographic studies of E. coli AspAT have revealed the binding site for the acidic substrate, but that for the hydrophobic substrate is still unknown. Site-directed mutagenesis of AspAT or AroAT has failed to reveal the hydrophobic pocket. Therefore, in order to search for the hydrophobic pocket, we constructed AspAT-AroAT chimeric proteins.
The chimeric proteins were obtained, even though the homology of the nucleotides was as low as 50%. The yield of chimeric gene was related to the length of homologous region between the two aminotransferase.The recombination occurred in the region where eight nucleotides out of ten were identical. It is suggested that the amino acid residues distant from active site contribute also contribute to the substrate specificity of aminotransferase.
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