| Project/Area Number |
09044228
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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 |
応用微生物学・応用生物化学
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| Research Institution | Yamaguchi University |
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Principal Investigator |
MATSUSHITA Kazunobu Faculty of Agriculture, Yamaguchi University, Professor, 農学部, 教授 (50107736)
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| Co-Investigator(Kenkyū-buntansha) |
MIGITA Taiko School of Allied Health Sciences, Yamaguchi University, Associate Professor, 医療技術短期大学部, 助教授 (90159161)
MIYOSHI Hideo Graduate school of Agriculture, Kyoto University, Associate Professor, 大学院・農学研究科, 助教授 (20190829)
YAMADA Mamoru Faculty of Agriculture, Yamaguchi University, Associate Professor, 農学部, 助教授 (30174741)
TAYAMA Hirohide Faculty of Agriculture, Yamaguchi University, Assistant Professor, 農学部, 助手 (60240884)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1997: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Pyrroloquinoline quinone / Quinoprotein dehydrogenase / Glucose dehydrogenase / Alcohol Dehydrogenase / X ray crystallography / Pseudomonas putida / Escherichia coli / Acetic acid bacteria |
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| Research Abstract |
Pyrroloquinoline quinone-dependent quinoprotein dehydrogenases, glucose dehydrogenase (GDH) of Escherichia coli and two alcohol dehydrogenases (ADH) of Pseudomonas putida and Gluconobacter suboxydans have been investigated to elucidate their structure and function, which was performed by several different means. 1)Structural and functional study of E. coli GDH : Several mutant enzymes were prepared by using random and site-directed mutageneses, and their reaction kinetics and oxido-reduction spectra were examined after purification. Compared with the structural model, the function of following amino acid residues, His-262, His-775, Trp-404, Asp-466, Asp-730 and Lys-493, have been identifisd. 2)Electron transfer study of P. putida ADH : (ADH IIB) was purified together with a blue copper protein, azurin, from the soluble fraction of P. putida and then the reaction between both proteins was examined by kinetic, fluorometric and redox titration analyses. As a result, the electron transfer between both proteins was shown to occur by a hydrophobic interaction and also by a freely reversible on and off binding process. Furthermore, the azurin-dependent alcohol oxidation activity could be reconstituted on the membrane vesicles and also with purified cytochrome oxidase. 3)Ubiquinone reaction site of E. coli GDH and G. suboxydans ADH : The ubiquinone-binding site of GDH was shown to be located near the surface of cytoplasmic membrane by reconstituting GDH with phospholipids having ubiquinone analogs at different depth. In G. suboxydans ADH, besides the ubiquinone reduction site, ubiquinol oxidation site was found to present separated from the site of ubiquinone reduction. 4)X ray crystallography of P. putida ADH and G. suboxydans ADH : The structure of ADH IIB of P. putida has already been successfully determined at 1.9 A. Whereas, G. suboxydans ADH has been crystallized and now under determination of the structure.
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