STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

恶臭假单胞菌的支链酮酸脱氢酶是由 在四种多肽E1 α、E1 β、E2和LPD-val中， 这个复合物的特定E3组分。 E1组件是 重要的催化作用，因为这是调节的网站， 恶臭假单胞菌和高等真核生物中的酶活性， 焦磷酸硫胺素 虽然E2和E3组件已被 虽然研究得很好，但对它的结构和功能知之甚少。 包括结合位点的酮酸脱氢酶E1组分 硫胺素焦磷酸盐 Bkd操纵子编码四个 该复合物的蛋白质已被分离，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E. coli建立基因顺序， 转录方向。 合并含有以下物质的提取物： 表达的蛋白质赋予酶活性，并为 分析每种成分。 最近发现上游地区 含有编码具有37.5%氨基酸的蛋白质的基因 与E.杆菌 证据来自含有该基因的质粒构建体， 体外诱变表明LRP样基因参与了 bkd操纵子的阳性表达。 甲基丙二酸半醛脱氢酶，在酶的最后一个酶， 缬氨酸分解代谢途径，在我们的实验室进行了表征 几年前，最近在哺乳动物中发现了这种基因。 的mms 含甲基丙二酸半醛脱氢酶操纵子 已从铜绿假单胞菌中克隆了一个结构基因并进行了序列测定 PAO。 该操纵子还包括以下基因： 3-羟基异丁酸脱氢酶，该酶立即 前甲基丙二酸半醛脱氢酶 分解代谢途径 一种基因，mmsR，编码一种调节蛋白 与AraC相似，直接位于上游。 失活 Escherichia coli也使其他两个基因失活，但突变体 仍然能够在缬氨酸琼脂上生长，表明存在替代途径 对缬氨酸代谢的影响。 本研究的具体目的是：1）研究结构 恶臭假单胞菌支链酮E1 α-β的功能 酸性脱氢酶2)研究LRP在人乳腺癌细胞表达中的作用， bkd操纵子。3)研究神经操纵子的调控。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.