Studies on Biochemical Functions of Pyrroloquinoline Quinone

藜麦蛋白脱氢酶根据其定位和性质可分为四类：(1)甲醇营养细菌中的甲醇脱氢酶；(2)以乙醇为唯一碳源生长的非甲基营养细菌中的藜麦蛋白醇脱氢酶；(3)在睾酮类丛生单胞菌中以脱辅酶形式形成藜麦蛋白乙醇脱氢酶，而以伯醇为碳源生长的恶臭假单胞菌则形成全酶。(4)藜麦血红蛋白乙醇脱氢酶-细胞色素c复合体在醋酸的细胞膜中形成。在本研究过程中，为了阐明吡咯喹啉醌和藜麦蛋白的功能，增加了以下新发现：(1)膜结合型、依赖于吡咯喹啉醌的乙醇脱氢酶是醋酸菌呼吸链中的主要脱氢酶。酶与…直接反应的能力在从乙酸乙酯和葡萄糖酸杆菌中分离纯化的乙醇脱氢酶中发现了更多的泛醌。乙醇氧化酶的重组是通过使用辛基葡萄糖苷洗涤剂透析法在室温下将无洗涤剂脱氢酶结合到预先从泛酚氧化酶和含有泛醌的磷脂中制备的蛋白脂质体上来完成的。重组的蛋白脂质体具有合理的乙醇氧化酶活性，其电子转移反应也能产生膜电位。(2)对甲氧基乙酸乙酸菌的甲醇和乙醇氧化酶呼吸链进行了研究。在本研究中，在另一种类型的甲醇脱氢酶中发现了一个32 KDa的多肽，它由三个不同的亚基组成，其中两个亚基分别对应于α亚基和β亚基。从土壤中分离鉴定的恶臭假单胞菌HK5产生三种不同的藜蛋白乙醇脱氢酶，其N端氨基酸序列与脱氮副球藻和甲基杆菌的moxJ基因高度同源，最终确定为甲醇氧化酶的MoxJ产物。当在乙醇上生长时，它会产生藜麦红蛋白酒精脱氢酶，而在甘油上生长，甘油是另一种可以氧化甘油的藜麦红蛋白酒精脱氢酶。第三种藜麦蛋白乙醇脱氢酶与在乙醇中生长的非甲基营养细菌中发现的类似。第三种乙醇脱氢酶不含血红素成分，不能以铁氰化钾为电子受体氧化乙醇。较少

The quinoprotein dehydrogenase has been classified into four groups according to their localization and characterization :(1) methanol dehydrogenase in methylotrophs,(2) quinoprotein alchold dehydrogenase from non-methylotrophic bacteria, when they are grown on ethanol as sole carbon source.(3) Quinohemoprotein alcohol dehydrogenase is formed as apo-enzyme in Commamonas testosteroni, while Pseudomonas putida forms holo-enzyme when they are grown on primary alcohols as the carbon source.(4) Quinohemoprotein alcohol dehydrogenase-cytochrome c complex is formed is formed in the cytoplasmic membranes of acetic acid bacteria.During the course of this research program, the following new findings have been added to clear up the function of pyrroloquinoline quinone and quinoproteins.(1) Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. An ability of the enzyme to directly react with … More ubiquinone was investigated in alcohol dehydrogenases purified from both Acetobacter aceti and Gluconobacter suboxydans. The reconstitution of alcohol oxidase was done by binding the detergent-free dehydrogenase at room temperature to proteoliposomes that had been prepared in advance from a ubiquinol oxidase and phospholipids containing ubiquinone by detergent dialysis using octyl-glucoside. The proteoliposomes thus reconstituted had a reasonable level of ethanol oxidase activity, the electron transfer reaction of which was also able to generate a membrane potential.(2) Methanol and ethanol oxidase respiratory chains of the methylotrophic acetic acid bacterium, Acetobacter methanolicus, was investigated. In this study, an 32 KDa peptide was found in alternative type of methanol dehydrogenase which contained three different subunits two of which corresponded to alpha-subunit and beta-subunit. The third subunit was finally concluded to be the MoxJ product in methanol oxidase, because the N-terminal amino acid sequence of the peptide showed a high homology to that of MoxJ gene from Paracoccus denitrificans or Methylobacterium extorquens AMl.(3) Pseudomonas putida HK5, isolated from soil and identified thereafter, produced three different quinoprotein alcohol dehydrogenase as holo-enzyme. When grown on ethanol, it produced a quinohemoprotein alcohol dehydrogenase, While grown on glycerol, another quinohemoprotein alcohol dehydrogenase which can oxidize glycerol. The third type of quinoprotein alcohol dehyderogenase was similar to those found in non-methylotrophic bacteria which were grown on ethanol. The third alcohol dehydrogenase contained no heme component and was not able to oxidize alcohol with potassium ferricyanide as an electron acceptor. Less