BIOCHEMISTRY AND GENETICS OF LIPOAMIDE DEHYDROGENASE

脂酰胺脱氢酶的生物化学和遗传学

• 批准号: 3278197
• 负责人: JOHN R SOKATCH
• 金额: $ 12.72万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278197
• 关键词: Pseudomonas; bacterial genetics; biochemical evolution; branched chain aminoacid; dihydrolipoamide dehydrogenase; enzyme complex; enzyme structure; flavoproteins; microorganism metabolism; molecular cloning; mutant; nucleic acid sequence; protein structure function; site directed mutagenesis; structural genes

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.

假单胞菌中存在三种lipoamide脱氢酶 提供了研究结构，功能和进化的独特机会 在氧化还原活性二硫键中。 LPD-VAL绝对具体 用于支链酮酸酸脱氢酶和LPD-GLC功能 丙酮酸和2-酮戊二酸脱氢酶。 第三个lipoamide 脱氢酶LPD-3可以替代丙酮酸中的LPD-GLC和 2-酮戊二酸脱氢酶复合物，但其主要功能是 未知。 这种情况提供了研究结构的机会 lipoamide脱氢酶的观点，以识别功能 域。 lipoamide脱氢酶的研究也具有医学上的重要性 由于该酶中的遗传缺陷是乳酸的原因之一 酸中毒是一种严重且通常是致命的遗传疾病。 这项研究的具体目的是： 1.确定LPD-3的作用。 这将通过 完成LPD3下游区域的核苷酸序列， 在LPD3中创建位于定向的突变，并研究诱导 p. putida的野生型和突变菌株中的LPD-3。 2.确定LPD-GLC的作用。 这将通过 通过创建LPDG上游的核苷酸序列 LPDG中的位置突变和通过LPDG的转录分析 表达。 3.确定LPD-VAL LPD-GLC和LPD-3的关系。 我们 将通过与P. p. p. p. putida菌株一起研究这一特定目标 LPDV，LPDG和LPD3中的位置定向突变，以查看这是什么影响 具有酶诱导和生长。 4。研究LPD-VAL的结构和功能。 W.G.J.霍尔在 格罗宁根大学将尝试结晶LPD-VAL。 知道 晶体结构，可以在 LPD-VAL的效果可以解释。 5.研究LPD-3 LPD-GLC和 LPD-VAL。 这将通过分析初级氨基酸来实现 这些蛋白质的序列使用Phylip程序。