Elucidation of Ubiquinone-binding Site of Membrane-bound Quinoproteins in Oxidative Bacteria

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This research project was performed by elucidating the ubiquinone-binding sites of membrane-bound quinoproteins of oxidative bacteria, glucose dehydrogenase (GDH) and alcohol dehydrogenase (ADH), which are functioning in the glucose oxidase respiratory chain of Escherichia coli and in the alcohol oxidase respiratory chain of acetic acid bacteria, respectively. GDH is a single peptide quinoprotein which consists of membrane-spanning N-terminal region and large soluble region containing prroloquinoline quinone (PQQ) and thus catalytic site for glucose. By screening specific inhibitors to prevent the reaction of GDH with ubiquinone, we have found several intense inhibitors of capsaicins analogs. Furthermore, we have examined their inhibitory mode for GDH and also the reaction site in the glucose oxidase respiratory chain. The inhibitor spectrum was also compared between GDH and mitochondrial NADH dehydrogenase, suggesting that GDH may have a structure for ubiquinone-binding site rather di … More fferent from NADH dehydrogenase. Now, we have been searching to isolate mutants of GDH insensitive to these inhibitors. Thereafter, we will figure our the amino acid residues and the structure involved in the ubiquinone-binding site by examining DNA sequence and kinetic analysis with mutated GDH.ADH,unlike GDH,is a quinohemoprotein which consists of three different subunits, subunit I containing PQQ and heme c, subunit II having 3 hemes c, and subunit III.By separating and then reconstituting the subunits of ADH,all three subunits have been shown to be required to have ubiquinone reductase activity, and the subunit II to have the ubiquinone-binding site. Furthermore, it has been shown that ADH has ubiquinol oxidase activity as well as ubiquinone reductase activity. By examining the effect of several ubiquinone analogs as the inhibitor and also the substrate, then both sites for ubiquinone oxidation and reduction was shown to have different structure and to be located in regions different from each other within subunit II. Less