1997 Fiscal Year Final Research Report Summary
Studies on enzymes and genes involved in biosynthesis of plant hormone, auxin
| Project/Area Number |
08456173
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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 |
Applied molecular and cellular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KOBAYASHI Michihiko Kyoto Univ.Agr.Senior Lecturer, 農学研究科, 講師 (70221976)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMIZU Sakayu Kyoto Univ.Agr.Prof., 農学研究科, 教授 (70093250)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Plant hormone / Indoleacetic acid / Nitrilase / Auxin / Gene |
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| Research Abstract |
Nitrilase in Alcaligenes faecalis, which forms the plant horomone indoleacetic acid from indole-36-acetonitrile, were analyzed at both protein and gene levels. The nitrilase was overexpressed in Escherichia coli. In order to investigate the carboxyl terminal region of this enzyme which is not homologous among various nitrilases so far known, we constructed a set of chimera enzymes between the A.faecalis nitrilase and Rhodococcus rhodochrous J1 (or R.rhodochrous K22). Each chimira enzyme was also overexpressed and showed similar substrate specificity to that of the wild A.faecalis nitrilase, suggesting the non-involvement of the carboxyl region of the enzyme in substrate specificity. The mutant enzyme that does not have the carboxyl region exhibited a very little nitrilase activity, indicating that this region would play an important role in the protein folding or the catalytic function.
On the other hand, the 1.5kb downstream from the nitrilase structural gene (nitA) of A.faecalis was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis using E.coli as a host. Sequence analysis of this region revealed the existence of an open reading frame (nitR) composing of 984 nucleotides. The deduced amino acid sequence of nitR showed similarity to that of a positive regulator family including XylS from Pseudomonas putida and AraC from E.coli. The nitR was found to code for a transcriptional positive regulator in nitA expression.
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Research Products
(14 results)
