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

5%的葡萄糖在尿酸循环中代谢，在这个循环中产生的木糖醇被认为与糖尿病并发症的发生有关。然而，产生和代谢木糖醇的酶的结构和性质还没有得到很好的研究。在这项研究中，我们对这个循环中的以下三种酶进行了表征，以阐明糖代谢以外的其他作用。L木糖还原酶(XR)。分离了人和4种啮齿动物XRS的基因，并对重组酶的性质进行了研究。哺乳动物的XRs是属于短链脱氢酶/还原酶家族的同源异构体，与双乙酰还原酶相同，能还原各种二羰基化合物，一些醛和酮。该酶广泛分布于哺乳动物的多种组织中，其中肝脏和肾脏组织中该酶含量较高。该酶定位于肾小管细胞刷状缘膜上。人的XR被结晶并确定了其晶体结构，…更多的是每个亚基都有一个分子内二硫键。此外，还通过定点突变，阐明了参与催化机制的3个残基、5个底物结合残基和冷失活的结构因素。通过筛选和基于晶体结构的计算机模拟，找到了一些XR的特异性和选择性的抑制剂，并用XR过表达的细胞测试了它们对细胞的通透性。结果：1.检测了XR基因转基因和基因敲除小鼠的生理效应。L-木糖醇脱氢酶。XDH在许多哺乳动物组织中高度分布于肝和肾中。对豚鼠肝脏胞浆和线粒体XDHs的纯化表明，它们是相同的，属于中链脱氢酶/还原酶家族的山梨醇脱氢酶。该酶氧化了几种芳香醇。L-古龙酸脱氢酶(GdH)。GDH的组织分布与XR、XDH相同。从兔肝中纯化的酶为二聚体，其氨基酸序列分析表明该酶属于3-羟基酰辅酶A脱氢酶家族。GDH接受几种无糖化合物作为底物。较少

项目成果

N.Tanaka., et al.: "SDR: Structure, Mechanism of Action, and Substrate Recognition"Current Organic Chemistry. 5. 89-111 (2001)

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.: "Enzymatic Characterization and Subcellular Distribution of a Short-chain Dehydrogenase/Reductase Family Protein, P26h, in Hamster Testis and Epididymis"Biochemistry. 130-132. 879-889 (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。