| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
There are different quinoprotein or quinohemoprotein alcohol dehydrogenases (ADH) in periplasm of pseudomonads and acetic acid bacteria, both of which function similarly as a primary dehydrogenase of the alcohol oxidase respiratory chain. However, both ADHs are different in terms of their localization and electron transfer reaction. In this study, the structure and function of type II ADH- and type III ADH-dependent electron transport systems of Pseudomonas putida and Gluconobacter suboxydans, respectively, was investigated comparatively.
1) Type II ADH of P. putida 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, flouorometric 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.
2) Furthermore, by reconstituting alcohol oxidase activity on the membrane vesicles and also with the purified cytochrome oxidase, cyanide-sensitive alcohol oxidase system was shown to be composed of ADH, azurin, and cytochrome oxidase. In addition, a cytochrome was shown to be present in the membrane and also to possibly be involved in cyanide-insensitive respiratory chain.
3) Type III ADH of G. suboxydans, besides ubiquinone reduction activity, was shown to have ubiquinol oxidation activity, which functions separated from the site of ubiquinone reduction. Furthermore, this ADH was also shown to be related to cyanide-insensitive ubiquinol oxidase activity.
4) Type II ADH of P. putida was crystallized and then the structure was successfully determined at 1.9 A with X ray crystallographic analysis. However, even though already crystallized, the structure of G. suboxydans ADH was not determined for the duration of this study.
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