STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

恶臭假单胞菌支链酮酸脱氢酶 在E1a、E1beta、E2和Lpd-Val四种多肽中， 这个复合体的特定E3组分。E1组件为 在催化中很重要，因为这是调节 恶臭毛滴虫和高等真核生物中的酶活性及其结合 焦磷酸硫胺素。尽管E2和E3组件已经 研究得很多，但对其结构和功能知之甚少 酮酸脱氢酶E1组分包括结合部位 用于硫胺素焦磷酸。编码四个基因的BKD操纵子 该复合体的蛋白质已被分离、测序并发现 包含四个紧密相连的基因。BKD基因已经被 亚克隆并在大肠杆菌中表达，建立基因序列 转录方向。组合包含 表达的蛋白质赋予酶活性，并为 对每种成分进行化验。最近，上游地区被发现 含有编码37.5%氨基酸的蛋白质的基因 与大肠杆菌亮氨酸反应蛋白(LRP)的同源性。 来自含有该基因的质粒构建的证据和在 体外诱变表明LRP样基因参与了 BKD操纵子阳性表达。 丙二酸甲酯半醛脱氢酶，最后一个酶 Valine分解代谢途径，在本实验室进行了表征 几年前，最近在哺乳动物身上发现了这种病毒。彩信 含有甲基丙二酸半醛脱氢酶的操纵子 铜绿假单胞菌结构基因的克隆及序列分析 鲍康如。MMS操纵子还包括 3-羟基异丁酸脱氢酶，该酶立即 缬氨酸中的甲基丙二酸半醛脱氢酶 分解代谢途径。编码一种调节蛋白的基因，MmsR 与AraC相似，紧随其后的是上游。停用 MMS也使另外两个基因失活，但突变体 仍然能够在Valine琼脂上生长，这表明有一种替代途径 用于缬氨酸新陈代谢。 本研究的具体目的是：1)研究结构 恶臭假单胞菌支链酮E1a-β的功能 酸脱氢酶。2)研究LRP在血管内皮生长因子表达中的作用 BKD操纵子。3)研究MMS操纵子的调控。4)寻找 铜绿假单胞菌中缬氨酸代谢的替代途径。

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.