UMP SYNTHASE AND THE MOLECULAR BASIS OF OROTIC ACIDURIA

UMP 合酶和乳清酸尿的分子基础

• 批准号: 3235252
• 负责人: DALE P SUTTLE
• 金额: $ 14.35万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-05-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235252
• 关键词: autosomal recessive trait; enzyme mechanism; fibroblasts; gene expression; genetic library; human tissue; ligase; messenger RNA; molecular pathology; nucleic acid probes; nucleic acid sequence; orotate phosphoribosyltransferase; orotic aciduria; orphan disease /drug; site directed mutagenesis; tissue /cell culture; transposon /insertion element; uridine monophosphate

UMP synthase is a bifunctional protein catalyzing the final two steps in the de novo biosynthesis of uridine 5' monophosphate (UMP). The two activities, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), catalyze the conversion of orotic acid to orotidine=5'-monophosphate (OMP) and OMP to UMP. As determined from the nucleotide sequence of the cDNA, UMP synthase is composed of 480 amino acids and has a molecular weight of 52,119. Hereditary orotic aciduria is a autosomal recessive disease associated with a severe deficiency of both activities of UMP synthase (Type I), or deficiency of only the ODC activity, (Type II). Orotic aciduria is the only specific disorder of de novo pyrimidine nucleotide biosynthesis described in humans. One of the primary objectives of this project is to identify the mutations in the structure of the UMP synthase protein associated with orotic aciduria. The sequence of the UMP synthase cDNA from orotic aciduria cells will be compared to the normal sequence. Sequence alterations identified in the mutant will be confirmed by various methods, including synthesis of the mutant protein from cDNA inserted into expression vectors, hybridization of allele specific oligonucleotides, or altered restriction site analysis. Another primary objective is to understand how the catalytic domains of UMP synthetase interact to form the normal bifunctional protein. In prokaryotes and lower eukaryotes, the two activities of UMP synthase reside in separate proteins. In mammals the two catalytic domains are joined by a linker or connector sequence. By changing the length and amino acid composition of the linker peptide we can determine the structural requirements for connecting the two catalytic domains. With the use of expression vectors we can also produce eukaryotic cells that synthesize separate proteins with OPRT and ODC activity or a single protein with the arrangement of the domains reversed. The stability and kinetic properties of the altered proteins will be compared to that of the normal bifunctional protein. Analysis of the defects in UMP synthase associated with orotic aciduria will increase our understanding of genetic defects and their effects on protein structure and activity. The characterization of the interaction of catalytic domains will provide information regarding enzyme evolution and cooperation, gone fusion, and substrate channeling.

UMP合酶是一种双功能蛋白，催化最后两个 尿苷 5' 单磷酸从头生物合成的步骤 （UMP）。 乳清酸磷酸核糖基转移酶的两种活性 (OPRT) 和乳清苷-5'-单磷酸脱羧酶 (ODC)，催化 乳清酸转化为乳清苷=5'-单磷酸（OMP） 和 OMP 到 UMP。 根据核苷酸序列确定 UMP合酶的cDNA由480个氨基酸组成，具有 分子量52,119。 遗传性乳清酸尿症是一种常染色体隐性遗传病 与 UMP 两种活性的严重缺乏有关 合成酶（I 型），或仅 ODC 活性缺陷（I 型） 二）。 乳清酸尿症是唯一特异的新发疾病 人类中描述的嘧啶核苷酸生物合成。 该项目的主要目标之一是确定 UMP合酶蛋白结构突变相关 伴有乳清酸尿症。 UMP 合酶 cDNA 的序列 乳清酸尿症细胞将与正常序列进行比较。 突变体中鉴定出的序列改变将通过 各种方法，包括从 cDNA插入表达载体，等位基因杂交 特定的寡核苷酸，或改变的限制性位点分析。 另一个主要目标是了解催化作用如何 UMP 合成酶的结构域相互作用形成正常的双功能 蛋白质。 在原核生物和低等真核生物中，这两种活性 的 UMP 合酶驻留在不同的蛋白质中。 在哺乳动物中，两者 催化结构域通过接头或连接器序列连接。 通过改变连接子的长度和氨基酸组成 肽我们可以确定连接的结构要求 两个催化域。 通过使用表达载体，我们 还可以产生合成单独蛋白质的真核细胞 具有 OPRT 和 ODC 活性或具有排列的单一蛋白质 域的反转。 稳定性和动力学特性 改变的蛋白质将与正常的蛋白质进行比较 双功能蛋白质。 乳腺相关UMP合酶缺陷分析 酸尿症将增加我们对遗传缺陷和 它们对蛋白质结构和活性的影响。 这 催化域相互作用的表征将 提供有关酶进化和合作的信息， 消失了融合和底物沟道。