Based on the deduced amino acid sequence of
Based on the deduced amino Nitrendipine sequence of the catalytic subunit of rG3DH, we could find sequence features that are highly conserved in FAD-harboring dehydrogenase, which include FAD binding motif and sequential cysteine residues in Cysteine-rich region. Several dehydrogenases share similarity of having hetero-oligomeric structure composed of a catalytic subunit containing FAD, a multiheme cytochrome complex subunit, and a chaperon-like subunit . From the primary structure resemblance between G3DHs and sorbitol dehydrogenase from Gluconobacter oxidans and 2-keto-d-gluconate dehydrogenases from Erwinia herbicola and Pantoea citrea , which are oligomeric enzymes made up of three subunits. The nucleotide sequencing of the gene fragment from Rhizobium sp. S10 also revealed three ORFs, therefore, we predict that G3DH is a three-component enzyme. However, the SDS-PAGE indicated that G3DH was expressed as a heteromeric enzyme consisting of only two subunits; the catalytic subunit (66 kDa) and the small subunit (18 kDa). An absent of the multiheme cytochrome c subunit might be because the aerobic cultivation affected the cytochrome c maturation in the host cells .
Characterization of purified rG3DH indicated that this enzyme shares the same range of the optimum pH and temperature with G3DHs from other strains . The highest G3DH activity could be detected at pH 7.0 and 40 °C. For substrate specificity, it is known that d-glucose shows the highest affinity to many G3DHs [6,, , ]. The rG3DH from Rhizobium sp. S10, however, showed better affinity toward disaccharides and glucose derivatives. This enzyme exhibited the highest activity toward cellobiose, which is the same for G3DHs from A. Tumefaciens  and F. Saccharophilum . Interestingly, the activity of the rG3DH slightly increased in the presence of FeSO4. This characteristic was observed in G3DH from A. Tumefaciens . The effect of FeSO4 might be explained by the existing of cysteine residues in Cysteine-rich region that form an iron-sulfur cluster and help electron transfer from FAD to cytochrome c .
Morimoto et al. established a novel process for mass production of d-gulose from an inexpensive lactose derivative. The highest keto-sugar conversion reported was 83%, while the maximum productivity was 0.0537 g/OD/L/h . Adopting their method with a few modifications, recombinant E. coli cells harboring the induced rG3DH could reach over 95% keto-sugar conversion rate and a relatively high productivity of 0.625 g/OD/L/h. Hence, the rG3DH from Rhizobium sp. S10 was effective for the keto-sugar production. The concentration of d-gulose in the reaction mixture should theoretically be 25%, yet the practical yield of d-gulose production was roughly 8%. The specific biological reduction and hydrogenation are desired for the better production of d-gulose.
To the best of our knowledge, G3DH has important functions in transport reaction as a member in energy-supplying systems and as a membrane structure maintenance effector . As well as in carbohydrate metabolism, G3DH converts glucosides to their corresponding 3-ketoglucosides, the first intermediate in the pathway [5,13]. Be able to react with the wide range of substrates, G3DH could provide potential applications in different fields such as sugar chemical industry, clinical diagnosis, and pharmaceutical intermediates synthesis [11,14]. Despite the importance of G3DH, the secondary-tertiary structure and the mechanism of G3DH are still unknown. Further research on genetic information is necessary to clarify the native structure of G3DH and its substrate binding mechanism.
Acknowledgements This study was supported in part by KAKENHI number 26450097, 22580088 (Grant-in-Aid for Scientific Research (C)).
Introduction Renal Cell Carcinoma (RCC) is one of the most common malignancies of the genitourinary tract, accounting for about 3% of all adult cancers . Despite advances in diagnosis, about 20–30% of all patients are diagnosed already with metastatic disease and extremely poor prognosis. Moreover one-third of RCC patients develops distant metastases after resection of the primary tumor . Tumor status has been associated with a number of metabolic and biochemical alterations. The subject of a new studies are certain molecules which could have a meaning in carcinogenesis and tumor progression. Several data have shown a role of tetraspanins, CD 133, calbindin, glutathione S-transferase P, retinal dehydrogenase 1 and aldehyde dehydrogenase in pathogenesis of renal cell carcinoma , , , .