BIOCHEMISTRY AND GENETICS OF LIPOAMIDE DEHYDROGENASE

假单胞菌中存在三种硫胺脱氢酶 为研究结构、功能和进化提供了独特的机会 在氧化还原活性的二硫键黄素蛋白中。LPD-VAL是绝对特定的 仅适用于支链酮酸脱氢酶和LPD-GLC功能 丙酮酸和2-酮戊二酸脱氢酶。第三种硫辛酰胺 脱氢酶LPD-3可以取代丙酮酸和丙酮酸中的LPD-GLC 2-酮戊二酸脱氢酶复合体，但其主要功能是 未知。这种情况为研究该结构提供了机会 硫酰胺脱氢酶，以期确定功能 域名。对硫酰胺脱氢酶的研究也具有重要的医学意义 由于这种酶的遗传缺陷是乳酸血症的原因之一 酸中毒，一种严重的、通常是致命的遗传病。 这项研究的具体目的是： 1.确定LPD-3的作用。这将通过以下方式实现 完成Lpd3下游区域的核苷酸序列，通过 在LPD3中产生定点突变，并通过研究 恶臭假单胞菌野生型和突变株的LPD-3。 2.确定LPD-GLC的作用。这将通过以下方式实现 完成lpdG上游的核苷酸序列，方法是 LpdG基因的定点突变及其转录分析 表情。 3.确定LPD-Val、LPD-GLC和LPD-3之间的关系。我们 我将通过创造一种恶臭假单胞菌菌株来研究这一特定目标 LpdV、lpdG和lpd3的定点突变，以了解这会产生什么影响 对酶的诱导和生长有抑制作用。 4.研究LPD-Val的结构和功能。W.G.J.霍尔 格罗宁根大学将尝试使LPD-Val具体化。了解 晶体结构，将有可能造成一系列突变 LPD-Val的影响可以解释。 5.研究LPD-3 LPD-GLC和LPD-GLC的进化关系 洛城警局-瓦尔。这将通过分析初级氨基酸来完成。 使用PHYLIP程序对这些蛋白质进行测序。

The existence of three lipoamide dehydrogenases in pseudomonads provides a unique opportunity to study structure, function and evolution in redox-active disulfide flavoproteins. LPD-val is absolutely specific for branched chain keto acid dehydrogenase and LPD-glc functions only with pyruvate and 2-ketoglutarate dehydrogenases. The third lipoamide dehydrogenase, LPD-3, can replace LPD-glc in the pyruvate and 2-ketoglutarate dehydrogenase complexes, but its primary function is unknown. This situation provides the opportunity to study the structure of lipoamide dehydrogenases with a view to identifying functional domains. Study of lipoamide dehydrogenases also has medical importance since genetic defects in this enzyme are one of the causes of lactic acidosis, a severe and usually fatal genetic disease. The Specific Aims of this research are to: 1.Determine the role of LPD-3. This will accomplished by completing the nucleotide sequence of the region downstream of lpd3, by creating site-directed mutations in lpd3 and by studying the induction of LPD-3 in both wild-type and mutant strains of P. putida. 2.Determine the role of LPD-glc. This will be accomplished by completing the nucleotide sequence upstream of lpdG, by creating site-directed mutations in lpdG and by a transcriptional analysis of lpdG expression. 3.Determine the relationships of LPD-val LPD-glc and LPD-3. We will study this specific aim by creating a strain of P. putida with site-directed mutations in lpdV, lpdG and lpd3 to see what effect this has on enzyme induction and growth. 4. Study the structure and function of LPD-val. W.G.J. Hol at the University of Groningen will attempt to crystallize LPD-val. Knowing the crystal structure, it will be possible to create a series of mutations in LPD-val whose effects can be interpreted. 5.Study the evolutionary relationships of LPD-3 LPD-glc and LPD-val. This will be accomplished by analyzing the primary amino acid sequences of these proteins using PHYLIP programs.