BIOCHEMISTRY AND GENETICS OF LIPOAMIDE DEHYDROGENASE

这项研究的长期目标是研究生物化学和 假单胞菌中硫辛酰胺脱氢酶的遗传学， 它们产生两种结构和功能不同硫辛酰胺 - 是的 哺乳动物产生一种硫辛酰胺脱氢酶， 提供丙酮酸、2-酮戊二酸和支链酮酸 脱氢酶。 在人类中，硫辛酰胺脱氢酶的遗传缺陷 导致新生儿乳酸性酸中毒，表现为快速 神经系统恶化和早逝 大肠杆菌产生一种 单一硫辛酰胺脱氢酶，其为丙酮酸的E3亚基， 2-酮戊二酸脱氢酶。 而恶臭假单胞菌（P. putida）和P. 铜绿假单胞菌产生两种硫辛酰胺脱氢酶，LPD-glc和LPD-val。 LPD-glc是生长过程中产生的唯一硫辛酰胺脱氢酶。 葡萄糖合成培养基，是2-酮戊二酸的E3亚基 脱氢酶和甘氨酸氧化中的L-因子。 生产LPD-val 在缬氨酸合成培养基中生长期间，是 支链酮酸脱氢酶 最近的数据显示， 也有一个单独的硫辛酰胺脱氢酶 恶臭假单胞菌中的脱氢酶。 E.大肠杆菌和猪心硫辛酰胺 还原酶、人谷胱甘肽还原酶和汞还原酶都是 氧化还原活性二硫化物黄素蛋白，并共享重要区域的 同源性表明这些显然是共同的进化途径， 不同的酶 基于氨基酸组成的研究表明， LPD-glc与E.大肠杆菌和猪心硫辛酰胺脱氢酶，但 而不是LPD-val。 LPD-val似乎与其他任何 氧化还原活性二硫化物黄素蛋白，除了可能汞还原酶。 本研究的具体目的是：1.确定一级结构 的LPD-val和LPD-glc。 这项研究的目的是 确定这两种蛋白质之间的进化关系， 其他氧化还原活性二硫化物黄素蛋白。 2.纯化丙酮酸 从恶臭假单胞菌脱氢酶，以确定是否有一个单独的结构 基因对于这个复杂的。

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.