|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2000 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1999 : ¥2,700,000 (Direct Cost : ¥2,700,000)
Dihydrodiol dehydrogenase (DD, EC 18.104.22.168), which catalyzes the NADP-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons, has been shown to be involved in cytotoxicity of the aromatic hydrocarbons. The enzyme exits in multiple forms in mammalian tissues. Of the multiple forms dimeric DD is tissue-specifically expressed, but its structure and physiological function have not been elucidated. This study was performed to elucidate the structure of dimeric DD, its relationship to the function and possible clinical significance. The results obtained are summarized.
1. Dimeric DD in monkey kidney, pig and dog liver was demonstrated to be identical with NADP-dependent D-xylose dehydrogenase (EC 22.214.171.124).
2. The protein sequencing and/or cDNA cloning primary structures of dimeric DDs in rabbit lens, human intestine and the above animal tissues revealed that the enzymes constitute a new protein family with prokaryotic proteins including glucose-fructose oxidoreductase.
3. The site
-directed mutagenesis on the monkey dimeric DD suggested that Tyr-180 is a catalytic residue, Lys-97 and His-79 function both coenzyme binding and chemical steps of the reaction, and Tyr-71 and His-76 are involved in coenzyme binding. In addition, Trp-125, Asp-176 and Trp-254 were suggested to be responsible for substrate binding, and of the three residues Asp-176 is thought to be important for the adaptation of the alcohol substrate in to the binding site. The crystallographic study is now in progress.
4. Dimeric DD was inhibited by several drugs such as nonsteroidal anti-inflammatory agents, of which the potent inhibitors commonly had 4-hydroxyphenylketone structure. The enzyme's mRNA, although it did not detected in normal human kidney, was detected in one of five renal cancer specimen. The variant gene with respect to exon 4 region was not detected, and such a study for other exon regions will be continued to find clinical significance.
5. During the course of cloning of the genomic gene for dimeric, I noticed that the gene is included in a working draft sequence of chromosome 19. The 5'-noncoding region of this gene contains various putative binding sites for transcription factors.
6. The structural and functional aspects obtained in this study was presented in 10^<th> International Symposium on Enzymology and Molecular Biology of Carbonyl Metabolism (New Mixico, U.S.A., July, 2000). Less