STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

支链酮酸脱氢酶的结构

• 批准号: 3227112
• 负责人: JOHN R SOKATCH
• 金额: $ 15.37万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227112
• 关键词: Escherichia coli; Pseudomonas; aldehyde /ketone oxidoreductase; aminoacid metabolism; bacterial genetics; chemical structure function; crystallization; enzyme activity; enzyme complex; enzyme structure; enzyme substrate complex; gene expression; ketoacid; leucine; maple syrup urine disease; microorganism culture; microorganism metabolism; molecular cloning; mutant; nucleic acid sequence; operon; orphan disease /drug; oxoglutarate dehydrogenase; protein sequence; pyruvates; radionuclides; regulatory gene; structural genes; valine

The branched chain keto acid dehydrogenase of P. putida is composed of four polypeptides, E1alpha, E1beta, E2 and LPD-val, the, specific E3 component of this complex. The E1 component is important in catalysis since this is the site of regulation of enzyme activity in both P. putida and higher eukaryotes and binds thiamin pyrophosphate. Although the E2 and E3 components have been well studied, little is known about the structure and function of keto acid dehydrogenase E1 components including the binding site for thiamin pyrophosphate. The bkd operon encoding the four proteins of the complex has been isolated, sequenced and found to contain four tightly linked genes. The bkd genes have been subcloned and expressed in E. coli establishing the gene order and direction of transcription. Combining extracts containing the expressed proteins gives enzyme activity and provides the basis for assays for each component. Recently, the upstream region was found to contain a gene encoding a protein with 37.5 % amino acid identity to the leucine responsive protein (LRP) of E. coli. Evidence from plasmid constructions containing this gene and in vitro mutagenesis indicate that the lrp-like gene is involved in the positive expression of the bkd operon. Methylmalonate semialdehyde dehydrogenase, the final enzyme in the valine catabolic pathway, was characterized in our laboratory several years ago and has recently been found in mammals. The mms operon containing the methylmalonate semialdehyde dehydrogenase structural gene has been cloned and sequenced from P. aeruginosa PAO. The mms operon also includes the gene for 3-hydroxyisobutyrate dehydrogenase, the enzyme immediately preceding methylmalonate semialdehyde dehydrogenase in the valine catabolic pathway. A gene, mmsR, encoding a regulatory protein with similarity to AraC, is immediately upstream. Inactivation of mms also inactivated the other two genes, but the mutants were still able to grow on valine agar suggesting an alternate pathway for valine metabolism. The specific aims of this research are to: 1) Study the structure and function of E1alpha-beta from P. putida branched chain keto acid dehydrogenase. 2) Study the action of LRP in expression of the bkd operon. 3) Study the regulation of the mms operon. 4) Look for alternate pathways of valine metabolism in P. aeruginosa.

p.utida的分支链酮酸脱氢酶组成 在四个多肽，e1alpha，e1beta，e2和lpd-val中， 该复合物的特定E3组件。 E1组件是 在催化中很重要，因为这是 P. p. p. p. p. p. p. and Excaryotes和较高的结合中的酶活性 硫胺素焦磷酸盐。 尽管E2和E3组件已经 研究良好，对 酮酸脱氢酶E1成分，包括结合位点 对于硫胺素焦磷酸盐。 编码四个的BKD操纵子 该复合物的蛋白质已被分离，测序并发现为 包含四个紧密链接的基因。 BKD基因已经 在大肠杆菌中取代并表达基因顺序和 转录方向。 结合包含的提取物 表达的蛋白质具有酶活性，并为 每个组件的测定。 最近，发现了上游地区 包含编码具有37.5％氨基酸的蛋白质的基因 大肠杆菌的亮氨酸反应蛋白（LRP）的身份。 来自包含该基因的质粒结构的证据 体外诱变表明LRP样基因参与 BKD操纵子的阳性表达。 甲基甲酸半二醛脱氢酶，最终酶 在我们的实验室中表征了Valine分解代谢途径 几年前，最近在哺乳动物中发现了。 MMS 含有甲基甲酸半醛脱氢酶的操纵子 结构基因已被克隆并从铜绿假单胞菌进行测序 pao。 MMS操纵子还包括 3-羟基异丁酸酯脱氢酶，酶立即 在缬氨酸中的甲基甲酸半甲醛脱氢酶之前 分解代谢途径。 编码调节蛋白的基因MMSR 与ARAC相似，立即上游。 失活 MM还灭活了其他两个基因，但突变体是 仍然能够在Valine琼脂上生长，建议替代途径 valine代谢。 这项研究的具体目的是：1）研究结构 P. p. putida分支链酮的e1alpha-beta的功能和功能 酸脱氢酶。 2）研究LRP在表达中的作用 BKD操纵子。 3）研究MMS操纵子的调节。 4）寻找 铜绿假单胞菌中缬氨酸代谢的替代途径。