MOLECULAR GENETICS OF ISOCITRATE DEHYDROGENASES

异柠檬酸脱氢酶的分子遗传学

• 批准号: 6741799
• 负责人: Lee McAlister-Henn
• 金额: $ 8.66万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6741799
• 关键词: Escherichia coli; Krebs' cycle; NAD(H) phosphate; allosteric site; biosynthesis; carbon; enzyme deficiency; enzyme mechanism; eukaryote; fungal genetics; gene expression; genetic strain; glucose 6 phosphate dehydrogenase; isocitrate dehydrogenase; isozymes; microorganism metabolism; mitochondria; molecular genetics; mutant; nucleic acid sequence; phenotype; polymerase chain reaction; yeasts

This research will use a molecular genetic approach to examine the regulation of a major intersection between catabolic and anabolic pathways in eucaryotic cells. Our focus is the family of isozymes of isocitrate dehydrogenase and our goal is to determine the role of these enzymes in controlling metabolic flux of carbon and reducing equivalents across compartmental barriers. These isozymes include a mitochondrial NAD+- specific enzyme (IDH), which catalyzes an allosterically regulated reaction in the tricarboxylic acid cycle, and two NADP+-specific enzymes, which are structurally similar but differentially localized in mitochondrial (IDP1) and cytosolic (IDP2) cellular compartments. Previous efforts have been focused on cloning and sequence analysis of the genes from Saccharomyces cerevisiae encoding each isozyme. Initial experiments in this proposal will examine the growth phenotypes and metabolic defects associated with loss of each isozyme by construction of mutant yeast strains containing all possible combinations of disruptions in genes encoding the isocitrate dehydrogenases. Subsequent experiments will test the importance of allosteric regulation and of mitochondrial localization for function of the NAD+-specific enzyme in the tricarboxylic acid cycle in vivo. This will be achieved by expression of mutant forms of that enzyme in yeast strains with defined metabolic defects. In other experiments, a collection of yeast mutants lacking combinations of the NADP+-specific isozymes and of a functional hexose monophosphate pathway will be constructed to assess relative contributions to cellular pools of biosynthetic NADPH reducing equivalents. Finally, these studies will be extended to mammalian systems with analyses of sequences and expression of cDNAs for the isocitrate dehydrogenases.

这项研究将使用分子遗传学方法来检查 调节分解代谢和合成代谢途径之间的主要交集 在桉树细胞中。 我们的重点是等酸的同工酶家族 脱氢酶和我们的目标是确定这些酶在 控制碳的代谢通量并减少跨 隔间障碍。 这些同工酶包括线粒体NAD+ - 特定的酶（IDH），催化了变构调节的 在三羧酸周期中的反应和两个NADP+特异性酶， 在结构上相似，但差异化 线粒体（IDP1）和胞质（IDP2）细胞室。 以前的 努力一直集中在基因的克隆和序列分析上 来自编码每个同工酶的酿酒酵母。 初始实验 在此提案中，将检查生长表型和代谢缺陷 通过构建突变酵母而与每种同工酶的损失相关 包含基因中断的所有可能组合的菌株 编码异位酸盐脱氢酶。 随后的实验将测试 变构调节和线粒体定位的重要性 用于三羧酸周期中NAD+特异性酶的功能 体内。这将通过表达突变形式来实现 酵母菌菌株中的酶，具有定义的代谢缺陷。 在其他 实验，缺乏组合的酵母突变体的集合 NADP+特异性同工酶和功能性己糖单磷酸途径 将构建以评估对细胞池的相对贡献 生物合成NADPH减少当量。最后，这些研究将是 扩展到具有分析序列和表达的哺乳动物系统 异位酸盐脱氢酶的cDNA。