2001 Fiscal Year Final Research Report Summary
Crystal structure and functional analysis of 2-oxacid : ferredoxin oxidoreductase
| Project/Area Number |
12660067
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | THE UNIVERSITY OF TOKYO |
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Principal Investigator |
WAKAGI Takayoshi Graduate School of Agriculture and Life Sciences, The University of Tokyo, Associate professor, 大学院・農学生命科学研究科, 助教授 (70175058)
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| Project Period (FY) |
2000 – 2001
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| Keywords | 2-oxoacid / ferredoxin / oxidoreductase / substrate specificity / thermophile / archaea / Sulfolobus / Aeropyrum |
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| Research Abstract |
2-Oxoacid : ferredoxin oxidoreductases (OFORs) from Sulfolobus sp. Strain 7 and from Aeropyrum were used for making crystals for X-ray crystallography. However, some of them formed crystals which were found to diffract X-ray with very low resolution.
Five possible active site residues that recognize 2-oxoacid in Sulfolobus OFOR were selected from the comparison of OFORs from Sulfolobus and other sources in terms of teriary structure and amino acid alignment. To eleuidate the role of these residues, they were subjected to site-directed mutagenesis to obtain a serious of variant enzymes. Arg344 and Thr353 of the subunit-a were essential and important, respectively. Lys49 and Leu123 of the subunit-b were responsible for the recognition of 2-oxoglutarate and pyruvate, respectively. A GXXGXG motif, supposed to bind to CoA, was examined. Each of the Gly was essential for enzyme activity, suggesting the importance of the motif.
During the course of OFOR research, an enzyme capable of catalyzing both decarboxylation and oxidation of indolepyruvate has been discovered. The reaction was independent of ferredoxin or CoA, and its final product was indoleacetic acid. The enzyme was a molybdo-flavo-iron-sulfur protein with biotin.
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