ENZYMES AND GENES OF P-AMINOBENZOATE BIOSYNTHESIS

对氨基苯甲酸生物合成的酶和基因

基本信息

批准号：
2182421
负责人：
BRIAN P NICHOLS
金额：
$ 13.98万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-04-01 至 1995-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2182421
关键词：
biochemical evolution dihydrofolate reductase enzyme biosynthesis gene mutation genetic library genetic transcription glutamine high performance liquid chromatography ligase lyase nuclear magnetic resonance spectroscopy nucleic acid sequence oligonucleotides p aminobenzoate protein structure site directed mutagenesis vitamin biosynthesis vitamin metabolism

项目摘要

rho-aminobenzoate (PABA) is an aromatic compound whose sole metabolic fate is its incorporation into the vitamin dihydrofolate. Derivatives of dihydrofolate participate in "one-carbon" metabolism of the cell, and are involved in the biosynthesis of precursors for DNA, protein, and lipids. Because folate is central to metabolism of macromolecules, the folate biosynthetic pathway has become the target for several antineoplastic, antifungal, antiprotozoal, and antibiotic agents. Additional knowledge concerning the biosynthesis of folate precursors may aid in the design and discovery similarly useful folate inhibitors. rho-aminobenzoate is synthesized from chorismate and glutamine in a two step pathway. rho- aminobenzoate synthase (PS) converts chorismate and glutamine to an intermediate, which is then converted to PABA by a newly discovered enzyme known only as "enzyme X". PS is a dimeric enzyme encoded by unlinked genes, pabA and pabB. Both pabA and pabB have been sequenced and are homologous with two other enzymes that use chorismate, anthranilate synthase and isochorismate synthase. In order to understand the evolutionary, structural, and metabolic relationships of these enzymes, this proposal seeks to characterize the enzymes of rho-aminobenzoate synthesis, and the expression of the genes that encode them. The identity of the intermediate in rho-aminobenzoate will be determined by isolation of the compound from enzyme reactions, followed by NMR analysis. The enzymes will be purified to homogeneity, and their kinetic parameters will be determined. Small molecule effectors of enzyme activity will also be investigated. pabA and pabB will be mutagenized using both oligonucleotide directed and chemical mutagenesis. Amino acid residues important in catalytic activity, protein structure, subunit interactions and feedback resistance will be identified. The expression of each of the genes encoding PS and enzyme X will be investigated. The genes are unlinked and apparently unregulated in response to rho-aminobenzoate or folate. Mechanisms that maintain constitutive, low level will be investigated by mutational analysis. Both site-directed mutagenesis using synthetic oligonucleotides, and chemical mutagenesis techniques will be used to construct and isolate mutations that affect expression of pabA, pabB, and the gene encoding enzyme X.

Rho-aminobenzoate（PABA）是一种芳香化合物，其唯一代谢命运它是掺入维生素二氢叶酸的。衍生物二氢叶酸参与细胞的“一碳”代谢，并且是参与DNA，蛋白质和脂质的前体的生物合成。因为叶酸是大分子代谢的核心，所以叶酸生物合成途径已成为几种抗肿瘤，抗真菌，抗体和抗生素剂。其他知识关于叶酸前体的生物合成可能有助于设计和发现类似地有用的叶酸抑制剂。 Rho-氨基苯甲酸盐是由依从酸盐和谷氨酰胺在两个步骤的途径中合成。 rh 氨基苯甲酸酯合酶（PS）将酸性和谷氨酰胺转化为中级，然后通过新发现的酶转化为PABA 仅称为“酶X”。 PS是由未链接编码的二聚体酶基因，PABA和PABB。 PABA和PABB都已测序，并且与另外两种使用合性，炭疽菌的酶同源合酶和异义素合酶。为了了解这些酶的进化，结构和代谢关系，该建议旨在表征Rho-aminobenzoate的酶综合，以及编码它们的基因的表达。身份 Rho-氨基苯甲酸酯中的中间体将通过分离来确定酶反应的化合物，然后进行NMR分析。酶将被纯化为同质性，它们的动力学参数将是决定。酶活性的小分子效应子也将是调查。 PABA和PABB将使用两种寡核苷酸进行诱变定向和化学诱变。氨基酸残基在催化活性，蛋白质结构，亚基相互作用和反馈将确定电阻。每个基因的表达将研究编码PS和酶X的编码。这些基因是未链接的，显然是针对Rho-氨基苯甲酸盐或叶酸不受管制的。维持组成型，低水平的机制将通过突变分析。两者使用合成的位置定向诱变寡核苷酸和化学诱变技术将用于影响PABA，PABB和编码酶X的基因。