2001 Fiscal Year Final Research Report Summary
Structure and function of the activator protein for Fe-type nitrile hydratase
| Project/Area Number |
12660093
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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 | Utsunomiya University (2001)
The Institute of Physical and Chemical Research (2000) |
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Principal Investigator |
ENDO Isao Utsunomiya University, Department of Agriculture, Professor, 農学部・生物生産科学科, 教授 (00087470)
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| Co-Investigator(Kenkyū-buntansha) |
YOHDA Masafumi Tokyo University of Agriculture and Technology, Department of Engineering, Associate Professor, 工学部, 助教授 (50250105)
ODAKA Masafumi RIKEN, Bioengineering Laboratory Senior Research Scientist, バイオ工学研究室, 先任研究員 (20224248)
UEDA Shunsaku Utsunomiya University, Department of Agriculture, Associate Professor, 農学部・生物生産科学科, 助教授 (80160167)
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| Project Period (FY) |
2000 – 2001
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| Keywords | cysteine-sulfinic acid / cystine-sulfenic acid / non-heme iron protein / post-translational modifications / nitrile hydratase / metallochaperone |
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| Research Abstract |
The Fe-type Nitrile hydratase (NHase) from Rhodococcus sp. N771 catalyzes the hydration of various nitriles to the corresponding amides, and consists of the ? and ? subunits with a unique non-heme iron catalytic center having two post-translationally modified cysteine ligands. An open reading frame, NHA3, was encoded at the downstream of the genes of the ? and ? subunits. We found that the co-expression of NHA3 was inevitable for the functional expression of the recombinant NHase in E. coli as well as in a Rhodococcus host-vector system. We named NHA3 as NHase activator. In the present research project, we studied the structure and function of the NHase activator.
1. Interaction between Fe-type NHase and the NHase activator. One or all of the cysteine residues in the potential metal-binding motif, ^<73>CXC^<77>C of NHase activator was substituted into serine by site-directed mutagenesis. The obtained mutant NHase activator proteins were co-expressed with the recombinant NHase in E. coli. In both cases, the yields of the NHase activity of the crude extracts were less than 15 % of that prepared from the E. coli. cells harboring the expression plasmid for native NHase activator. The result suggests NHase activator is involved in the insertion of an iron ion into the metallocenter of NHase. We also constructed the expression vector for His-tagged NHase activator. His-tagged NHase activator was co-expressed with the ? and ? subunits of NHase, and subjected into Ni-NTA agarose affinity column. The eluate was analyzed by western-blotting. It was found that the ? and ? subunits eluted together with NHase activator. Thus, it was suggested that NHase activator interacted with the the ? and ? subunits of NHase.
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