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Comparative and Biochemical Studies on Periplasmic Alcohol Oxidase Systems in Pseudomonads and Acetic Acid Bacteria

假单胞菌和乙酸菌周质醇氧化酶系统的比较和生化研究

基本信息

批准号：
10660091
负责人：
MATSUSHITA Kazunobu
金额：
$ 2.24万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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.

在假单胞菌和醋酸菌的周质中存在不同的醌蛋白或醌血红素蛋白醇脱氢酶（ADH），它们都作为醇氧化酶呼吸链的初级脱氢酶起类似的作用。然而，这两种ADH在它们的定位和电子转移反应方面是不同的。本研究比较研究了恶臭假单胞菌（Pseudomonas putida）和亚氧化葡糖酸杆菌（Gluconecrosporsuboxanans）的Ⅱ型ADH和Ⅲ型ADH依赖的电子传递系统的结构和功能。1）从恶臭假单胞菌的可溶性组分中分离纯化了Ⅱ型ADH和蓝铜蛋白azurin，荧光和氧化还原滴定分析。结果表明，这两种蛋白质之间的电子传递是通过疏水相互作用和自由可逆的开-关结合过程进行的。2）此外，通过在膜囊泡上和纯化的细胞色素氧化酶上重建醇氧化酶活性，氰化物敏感的醇氧化酶系统由ADH、天青蛋白和细胞色素氧化酶组成。此外，细胞色素被证明是存在于膜上，也可能参与氰化物不敏感的呼吸链。除了泛醌还原活性外，还显示出suboxetans具有泛醇氧化活性，其功能与泛醌还原位点分离。此外，该ADH还与氰化物不敏感的泛醇氧化酶活性有关。4）对恶臭假单胞菌的II型ADH进行了结晶，并在1.9 A下用X射线晶体学分析成功地确定了其结构。然而，即使已经结晶，G.在本研究期间，未测定suboxanans ADH。