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

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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