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

5%的葡萄糖在糖醛酸循环中代谢，在此循环中产生的木糖醇被认为与糖尿病并发症的发展有关。然而，生产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，我们描述了这个循环的以下三种酶，以阐明糖代谢以外的作用。L-木酮糖还原酶（XR）。分离了人和四种啮齿动物XRs的cDNA，并对重组酶的性质进行了研究。哺乳动物XR是属于短链脱氢酶/还原酶家族的同源四聚体，与双乙酰还原酶相同，并还原各种二羰基化合物，一些醛和酮。该酶在哺乳动物的多种组织中均有分布，其中肝脏和肾脏中的含量较高。该酶定位于肾小管细胞刷状缘膜上。将人XR结晶并确定其晶体结构， ...更多信息 每个亚基都有一个分子内二硫键。此外，还通过定点突变的方法确定了3个参与催化机制的残基、5个与底物结合的残基和冷失活的结构因子。通过筛选和基于晶体结构的计算机模拟，发现了一些特异性和选择性的XR抑制剂，并使用XR过表达的细胞测试了它们对细胞的渗透性。检测XR基因转基因和敲除小鼠的生理效应. L-木糖醇脱氢酶（XDH）。XDH在多种哺乳动物的肝、肾组织中有较高的分布。纯化豚鼠肝脏的胞质和线粒体XDH显示它们是相同的，并且是属于中链脱氢酶/还原酶家族的山梨醇脱氢酶。该酶能氧化多种芳香醇. L-古洛酸脱氢酶（GDH）。GDH的组织分布也与XR和XDH相同。从兔肝中纯化的酶为二聚体，氨基酸序列分析表明该酶属于3-羟酰辅酶A脱氢酶家族。GDH接受几种无糖化合物作为底物。少

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