1992 Fiscal Year Final Research Report Summary
Protein Engineering of Aspartate Aminotransferase
| 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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| Keywords | Enzyme / Catalytic Mechanism / Substrate Recogition / Protein Engineering / Aminotransferase / Chimera / X-Ray Crystallography / Site-Directed Mutagenesis |
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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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[Publications] KYRAMITSU,S.: "Asp222,His143,and Tyr70 in Escherichia coli Aspartate Aminotransferase" ″Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors″(Fukui,T.,Kagamiyama,H.,Soda,K.,& Wada,H., eds.) Rergamon Press,Oxford. 179-181 (1991)
Description
「研究成果報告書概要(和文)」より
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[Publications] HAYASHI,H.: "Escherichia coli Aspartate Aminotransferase: Effects of Site-Specific Mutationson Substrate Binding and Catalysis" ″Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors″(Fukui,T.,Kagamiyama,H.,Soda,K.,& Wada,H.,eds.) Pergamon Press,Oxford. 183-186 (1991)
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「研究成果報告書概要(和文)」より
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[Publications] Sung,M.-H.: "Molecular Cloning and Sequence Determination of Thermostable Aspartate Aminotransferase from Thermophilic Bacillus Species" ″Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors″(Fukui,T.,Kagamiyama,H.,Soda,K.,& Wada,H.,eds.) Pergamon Press,OXford. 107-109 (1991)
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「研究成果報告書概要(和文)」より
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[Publications] Inoue,K., Kuramitsu,S., Okamoto,A., Hirotsu,K., Higuchi,T., and Kagamiyama,H: "Site-Directed Mutagenesis of Escherichia coli Aspartate Aminotransferase: Role of Tyr70 in the Catalytic Processes" Biochemistry. 30(No.30). 7796-7801 (1991)
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「研究成果報告書概要(欧文)」より
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[Publications] Kuramitsu,S., Yano,T., Inoue,K., Hiromi,K., Tanase,S., Morino, Y., and Kagamiyama,H: "Asp222, His143, and Tyr70 in Escherichia coli Aspartate Aminotransferase" "Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors"(Fukui,T., Kagamiyama,H., Soda,K., and Wada,H., eds.), Pergamon Press,Oxford. 179-181 (1991)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Hayashi,H., Inoue,Y., Kuramitsu,S., and Kagamiyama,H: "Escherichia coli Aspartate Aminotransferase: Effects of Site-specific Mutationson on Substrate Binding and Catalysis" "Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors"(Fukui,T., Kagamiyama,H., Soda,K., and Wada,H., eds.), Pergamon Press,Oxford. 183-186 (1991)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Sung,M.-H., Tanizawa,K., Tanaka,H., Kuramitsu,S., Kagamiyama,H., and Soda,K: "Molecular Cloning and Sequence Determination of Thermostable Aspartate Aminotransferase from Thermophilic Bacillus Species" "Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors"(Fukui,T.,Kagamiyama,H., Soda,K., and Wada,H., eds.), Pergamon Press, Oxford. 107-109 (1991)
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「研究成果報告書概要(欧文)」より
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[Publications] Okamoto,A., Hirotsu,K., Higuchi,T., Kuramitsu,S., and Kagamiyama,H: "Three-Dimensional Structure of Aspartate Aminotransferase from Escherichia coli" "Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors"(Fukui,T., Kagamiyama,H., Soda,K., and Wada,H., eds.), Pergamon Press,Oxford. 55-58 (1991)
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[Publications] Tanaka,T., Yamanoto,S., Taniguchi,M., Hayashi,H., Kuramitsu,S., Kagamiyama,H., and Oi,S: "Further Studies on Aspartate Aminotransferase of Thermophilic Methanogens by Analysis of General Properties, Bound Cofactors, and Subunite Structures" J.Biochem. 112(No.6). 811-815 (1992)
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「研究成果報告書概要(欧文)」より
