| Project/Area Number |
12440191
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Inorganic chemistry
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| Research Institution | RIKEN |
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Principal Investigator |
ODAKA Masafumi RIKEN, Bioengineering Laboratory, Senior Research Scientist, バイオ学研究室, 先任研究員 (20224248)
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| Co-Investigator(Kenkyū-buntansha) |
KATAYAMA Yoko Tokyo University of Agriculture and Technology, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (90165415)
YOHDA Masafumi Tokyo University of Agriculture and Technology, Faculty of Engineering, Associate Professor, 工学部, 助教授 (50250105)
ENDO Isao Utsunomiya University, Department of Agriculture, Professor, 農学部, 教授 (00087470)
NYUNOYA Hiroshi Tokyo University of Agriculture and Technology, Gene Research Center, Professor, 遺伝子実験施設, 助教授 (60135936)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | cysteine-sulfinic acid / cysteine-sulfenic acid / non-heme protein / post-translational modification / nitrile hydratase / thiocyanate hydrolase / metallochapeorne / non-corrin cobalt protein / 非ヘム鉄蛋白質 / 非ヘム鉄タンパク質 |
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| Research Abstract |
Nitrile hydratases (NHase) are metalloenzymes containing a non-heme or a non-corrin cobalt active center and catalyze the hydration of various nitriles to the corresponding amides. We have shown that Fe-type NHase of Rhodococcus sp. N771 has a novel metal-binding motif containing two oxidized cysteine ligands, cysteine-sulfenic acid (Cys-SOH) and cysteine-sulfinic acid (Cys-SO2H). In the present research, we studied functions of the novel metal-binding motif in nitrile hydratase family.
1.Function of the oxidized cysteine ligands-Isobutyronitrile (IBN) had been reported as a competitive inhibitor with a Ki value of 5 μM. We found that authentic IBN was hydrated normally and that the impurity present in commercially available IBN, 2-cyano-2-propyl hydroperoxide (Cpx) inhibited NHase activity strongly. Cpx specifically oxidized the Cys-SOH ligand in the metal-binding motif, to inactivate the enzyme irreversibly n-Butyric acid (BA) is known to a stabilizing agent of NHase and used for purification, storage and most experiments reported. We studied how BA stabilizes NHase. We showed that BA protected αCys114-SOH from aerobic oxidation to Cys-SO_2H. Both results results demonstrated that the Cys-SOH structure of αCys114 is essential for the catalytic activity.
2.We revealed that the Cys-SO2H modification was conserved not only in Co-type NHase but also in thiocyanate hydrolase (SCNase) whose amino acid sequences were well conserved with NHases. We also have shown that SCNase is a novel member of Co-type NHase. We constructed the expression system of apo-SCNase in E..coli, and succeeded in its crystallization. X-ray crystal structure analysis of apo-SCNase is currently underway.
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