|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
Five percent of glucose metabolized in the uronate cycle, xylitol produced in this cycle has been suggested to be involved in development of diabetic complications. However, the structure and properties of enzymes that produce and metabolize xylitol have not been well studied. In this study, we characterized the following three enzymes of this cycle to elucidate roles other than sugar metabolism.
1. L-Xylulose reductase (XR). The cDNAs for XRs of human and four rodents were isolated, and the properties of the recombinant enzymes were studied. Mammalian XRs are homotetramers belonging to the short-chain dehydrogenase/reductase family, are identical to diacetyl reductase, and reduce various dicarbonyl compounds, some aldehydes and ketones. The enzyme was distributed in many mammalian tissues, in which liver and kidney show high contents of the enzyme. The enzyme was localized on brush-border membranes of renal tubular cells. Human XR was crystallized and determined its crystal structure,
which had a intramolecular disulfide bond per subunit. In addition, three residues involving in the catalytic mechanism, five substrate-binding residues and structural factors of cold inactivation were elucidated by site-directed mutagenesis. Some specific and selective inhibitors of XR were found by screening and computer-modeling based on the crystal structure, and their permeability into the cell was tested using XR-overexpressed cells. Physiological effects of the XR-gene transgenic and knockout mice were examined.
2. L-Xylitol dehydrogenase (XDH). XDH was highly distributed in liver and kidney of many mammalian tissues. Purification of cytosolic and mitochondrial XDHs of guinea pig liver revealed that they are identical and are sorbitol dehydrogenase that belongs to the medium-chain dehydrogenase/reductase family. The enzyme oxidized several aromatic alcohols.
3. L-Gulonate dehydrogenase (GDH). The tissue distribution of GDH was also the same as those of XR and XDH. The enzymes purified from rabbit liver was dimeric, and its amino acid sequence analysis suggested that this enzyme belongs to the 3-hydroxyacyl CoA dehydrogenase family. GDH accepts several no-sugar compounds as the substrates. Less