BIOCHEMISTRY AND GENETICS OF LIPOAMIDE DEHYDROGENASE

这项研究的长期目标是研究生物化学和 假单胞菌中硫酰胺脱氢酶的遗传学 它们会产生两种结构和功能不同的硫酰胺 脱氢酶。哺乳动物产生单一的硫酰胺脱氢酶，该酶 供应丙酮酸、2-酮戊二酸和支链酮酸 脱氢酶。在人类中，硫酰胺脱氢酶基因缺陷 结果导致新生儿乳酸酸中毒，表现为快速 神经退化和过早死亡。大肠埃希菌产生一种 单硫酰胺脱氢酶，是丙酮酸的E3亚基和 2-酮戊二酸脱氢酶。相比之下，恶臭假单胞菌和恶臭假单胞菌均有不同程度的毒力。 铜绿假单胞菌产生两种硫胺脱氢酶，LPD-GLC和LPD-Val。 LPD-GLC是植物生长过程中唯一产生的硫胺脱氢酶。 葡萄糖合成介质，是2-酮戊二酸的E3亚单位 脱氢酶与甘氨酸氧化中的L因子。产生了LPD-VAL 在Valine合成培养基中生长过程中，它是E3亚基 支链酮酸脱氢酶。最近的数据增加了这种可能性 丙酮酸还有一个单独的硫胺脱氢酶 恶臭假单胞菌脱氢酶。大肠埃希菌和猪心硫酰胺 脱氢酶、人谷胱甘肽还原酶和汞还原酶都是 氧化还原活性的二硫代黄素蛋白，并共享重要区域 同源性表明这些显然是一条共同的进化路径 完全不同的酶。基于氨基酸组成的研究表明 LPD-GLC与大肠杆菌和猪心硫酰胺脱氢酶有关，但 不是给LPD-Val的。LPD-Val似乎与任何其他 氧化还原活性的二硫代黄素蛋白，可能的汞还原酶除外。 本研究的具体目的是：1.确定一级结构 LPD-Val和LPD-GLC。这项研究的目标将是 确定这两种蛋白质之间的进化关系 其他具有氧化还原活性的二硫键黄素蛋白。2.提纯丙酮酸 从恶臭假单胞菌中分离脱氢酶以确定是否存在单独的结构 这个复合体的基因。

The long term objective of this research is to study the biochemistry and genetics of lipoamide dehydrogenases in pseudomonads which are unique in that they produce two structurally and functionally distinct lipoamide dehydrogenases. Mammals produce a single lipoamide dehydrogenase which serves pyruvate, 2-ketoglutarate and branched chain keto acid dehydrogenase. In man, genetic deficiency of lipoamide dehydrogenase results in lactic acidosis in newborns which is manifested by rapid neurological deterioration and an early death. Escherichia coli produces a single lipoamide dehydrogenase which is the E3 subunit of pyruvate and 2-ketoglutarate dehydrogenases. In contrast, both P. putida and P. aeruginosa produce two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-glc is the only lipoamide dehydrogenase produce during growth on glucose synthetic medium and is the E3 subunit of 2-ketoglutarate dehydrogenase and the L-factor in glycine oxidation. LPD-val is produced during growth in valine synthetic medium and is the specific E3 subunit of branched chain keto acid dehydrogenase. Recent data raised the possibility that there is also a separate lipoamide dehydrogenase for pyruvate dehydrogenase in P. putida. E. coli and pig heart lipoamide dehydrogenases, human glutathione reductase and mercuric reductase are all redox-active disulfide flavoproteins and share significant regions of homology suggesting a common evolutionary pathway for these apparently disparate enzymes. Studies based on amino acid compositions suggest the LPD-glc is related to E. coli and pig heart lipoamide dehydrogenases, but not to LPD-val. LPD-val appears to be unrelated to any of the other redox-active disulfide flavoproteins, except possibly mercuric reductase. The Specific Aims of this study are to 1: Determine the primary structure of LPD-val and LPD-glc. The objective of this research will be to determine the evolutionary relationships between these two proteins and other redox-active disulfide flavoproteins. 2. Purify pyruvate dehydrogenase from P. putida to determine if there is a separate structural gene for this complex.