| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
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
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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
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