Study on the uronate-cycle oxidoreductases that are expressed highly in kidney

肾脏高表达糖醛酸循环氧化还原酶的研究

• 批准号: 13672290
• 负责人: HARA Akira
• 金额: $ 2.3万
• 依托单位: Gifu Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672290/
• 关键词: L-xylulose reductase; crystal structure; uranate cycle; xylitol dehydrogenase; L-gulonate dehydrogenase; drug metabolism; SDRファミリー; ジアセチル還元酵素; 結晶化; イサチン

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, … More 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

在uronate循环中代谢的葡萄糖中有5％，在此周期中产生的木糖醇被认为参与糖尿病并发症的发展。但是，尚未对产生和代谢木糖醇的酶的结构和特性进行很好的研究。在这项研究中，我们表征了该周期的以下三种酶，以阐明糖代谢以外的其他作用。1。 L-二硫次还原酶（XR）。分离了人类和四个啮齿动物的XR的cDNA，重组酶的特性进行了研究。哺乳动物XR是属于短链脱氢酶/还原酶家族的同二探测器，与二乙酰还还原酶相同，并减少各种二烷基化合物，一些醛和酮。该酶分布在许多哺乳动物组织中，其中肝脏和肾脏表现出酶的含量很高。该酶位于肾结结核病的刷子机制上。人XR被结晶并确定其晶体结构，…更多，每个亚基具有分子内二硫键。此外，通过位置定向的诱变阐明了催化机理中涉及的三个残留物，五个底物结合抗性和冷灭活的结构因子。通过基于晶体结构进行筛选和计算机模型，发现了一些特异性和选择性抑制剂，并使用XR越过表达的细胞测试了它们对细胞的渗透性。检查了XR基因转基因和基因敲除小鼠的生理效应2。 L-木糖醇脱氢酶（XDH）。 XDH高度分布在许多哺乳动物组织的肝脏和肾脏中。豚鼠肝脏的胞质和线粒体XDHS的纯化表明它们是相同的，并且是属于中链脱氢酶/还原酶家族的山梨糖醇脱氢酶。酶氧化了几种芳香醇。3。 L-硫酸盐脱氢酶（GDH）。 GDH的组织分布也与XR和XDH的组织分布相同。从兔肝脏纯化的酶是二聚体，其氨基酸序列分析表明该酶属于3-羟基乙酰乙酰基COA脱氢酶家族。 GDH接受几种无糖化合物作为底物。较少的

项目成果

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N.Tanaka. 等人：“SDR：结构、作用机制和底物识别”当前有机化学。

S.Ishikura., et al.: "Molecular Cloning of a cDNA for Hamster Diacetyl Reductase that Is Identical with L-Xylulose Reductase"Chemico-Biological Interactions. 130-132,879-889 (2001)

S.Ishikura.等人：“与L-木酮糖还原酶相同的仓鼠二乙酰还原酶的cDNA的分子克隆”化学-生物相互作用。

S.Ishikura., et al.: "Enzymatic Characterization and Subcellular Distribution of a Short-chain Dehydrogenase/Reductase Family Protein, P26h, in Hamster Testis and Epididymis"Biochemistry. 40. 214-224 (2001)

S.Ishikura.等人：“仓鼠睾丸和附睾中短链脱氢酶/还原酶家族蛋白 P26h 的酶学特征和亚细胞分布”生物化学。

S.Ishikura., et al.: "Identification of Amino Acid Residues Involved in Substrate Recognition of L-Xylulose Reductase by Site-Directed Mutagenesis"Chemico-Biological Interactions. 143-144,543-550 (2003)

S.Ishikura.等人：“通过定点诱变鉴定参与L-木酮糖还原酶底物识别的氨基酸残基”化学-生物相互作用。

V.Carbone, C., et al.: "Structure-Basee Design of Inhibitors of Human L-Xylulose Reductase Modelled into the Active-Site of the Enzyme"Bioorg. Med. Chem. Lett..

V.Carbone，C.等人：“基于结构的人L-木酮糖还原酶抑制剂的设计，建模为酶的活性位点”Bioorg。