Quinoprotein Glycerol Dehydrogenase of Acetic Acid Bacteria and 5-Ketogluconate Production

醋酸菌胞质膜上的奎诺蛋白甘油脱氢酶（GLDH）以PQQ为假体基，不仅能氧化多醇，还能氧化d -葡萄糖酸盐，反应产物为5-酮葡萄糖酸盐（5-KGA）。在这项研究中，我们研究了独特的底物特异性，以及金属或吡咯醌醌（PQQ）的结合方式。此外，我们还试图通过基因工程的方法创造出一个高5- kga产量的菌株。金属与PQQ的结合方式：edta处理后的纯化酶对辅因子的需求表明，Mg^<2+>对与PQQ形成全酶起关键作用。Ca^<2+>进一步刺激Mg^<2+>-活化酶，使酶活性达到最高，这一现象在其他藜蛋白中从未见过。利用GLDH的荧光猝灭法对全酶形成过程中的辅助因子进行了滴定。通过滴定获得的结合动力学显示两个PQQ-和两个M…更多的g^<2+>-结合位点和一个Ca^<2+>-结合位点。此外，还表明纯化后的酶在某些洗涤剂的存在下以二聚体形式存在。因此，认为二聚化反应与GLDH.2的两个催化位点之间存在一定的关系。GLDH的催化位点：当酶在Mg^<2+>全异构化时，GLDH具有另外一个独特的性质，即在酸性和碱性ph值两个最佳ph值。而仅用Ca^<2+>制备全酶时，GLDH只有酸性pH值最优。除d -葡萄糖酸盐外，在任何GLDH底物上都可以观察到这些活性。与“体内”系统不同，当底物为d -葡萄糖酸盐时，膜组分和纯化形式的GLDH在酸性pH下表现出相对较弱的活性，而在碱性pH下则没有活性。抗d -葡萄糖酸盐活性降低的原因被证明是由于在碱性pH下与d -葡萄糖酸解离形式的金属螯合作用，以及在酸性pH下内酯的形成。高产5-KGA菌株的产生：在两株葡萄球菌中，干扰5-KGA生产的2-酮葡萄糖酸产生酶被破坏。其中一种破坏菌是唯一一种产生5kga的菌株。现在，我们还尝试进行呼吸链突变和/或内酯降解酶突变，以获得更高的5- kga生产者。x射线晶体学和催化位点的修饰：我们一直试图通过改变许多不同的洗涤剂来得到GLDH的晶体，但目前我们还不能成功地得到一个很好的晶体。而且，由于我们可以为GLDH突变体创建大肠杆菌表达系统，现在我们开始做容易出错的PCR来获得一些催化位点突变体。少

Quinoprotein, glycerol dehydrogenase (GLDH), on the cytoplasmic membranes of acetic acid bacteria has PQQ as the prosthetic group and oxidizes not only polyalcohols but also D-gluconate, of which the reaction product is 5-ketogluconate (5-KGA). In this study, we have examined the unique substrate specificity, and also the binding mode of metal or pyrroloquinone quinone (PQQ). Furthermore, we have also tried to create a high 5-KGA-producing strain by means of genetic engineering.1.Binding mode of metal and PQQ : The cofactor requirements of EDTA-treated purified enzyme indicated that Mg^<2+> plays a key role to form holo-enzyme with PQQ. Ca^<2+> stimulated further the Mg^<2+>-activated enzyme to exhibit the maximum enzyme activity, of which the phenomenon has never been seen in any other quinoproteins. Titration of cofactor during the holo-enzyme formation were performed by using the fluorescence quenching of GLDH. Binding kinetics obtained by the titrations exhibited two PQQ- and two M … More g^<2+>-binding sites and one Ca^<2+>-binding site. In addition, it was shown that the purified enzyme exists as a dimer form in the presence of some detergent. Thus, it is suggested that there is some relations between the dimerization and the two catalytic sites of GLDH.2.Catalytic site of GLDH : GLDH has an additional unique property, two optimum pHs, both at acidic and alkaline pHs when the enzyme was holomerized with Mg^<2+>. Whereas, GLDH exhibited only acidic pH optimum when holo-enzyme was prepared only with Ca^<2+>. These activities observed at both pHs were observed with any substrate for GLDH, except for D-gluconate. Unlike in the "in vivo" system, GLDH in the membrane fraction and also in the purified form exhibited a relatively weak activity at acidic pH and no activity at alkaline pH when substrate is D-gluconate. The reason for these decreased activity against D-gluconate was shown to be due to metal-chelating with dissociated form of D-gluconate at alkaline pH, and also due to lactone formation at acidic pH.3.Creation of high 5-KGA-producing strain : 2-Ketogluconate producing enzyme which may disturb 5-KGA production was disrupted in two strains of Gluconobacter. One of the disruptants turned out to be sole 5-KGA-producing strain. Now, we have also tried to make a respiratory chain mutation and/or lactone-degrading enzyme mutation to get a higher 5-KGA-producer.4.X-ray crystallography and modification of the catalytic site : We have been trying to get a crystal of GLDH by changing many different detergents, but at this moment we could not be succeeded to get a nice crystal. And, since we could create E.coli expression system for GLDH mutants, now we are starting to do error-prone PCR to get some catalytic site mutants. Less