ENZYMES AND GENES OF P-AMINOBENZOATE BIOSYNTHESIS

Rho-氨基苯甲酸酯(PABA)是一种芳香族化合物，其唯一的代谢命运 就是它与维他命二氢叶酸的结合。衍生品 二氢叶酸参与细胞的“一碳”代谢，并 参与DNA、蛋白质和脂肪前体的生物合成的。 因为叶酸是大分子新陈代谢的中心，所以叶酸 生物合成途径已成为几种抗肿瘤药物的靶点， 抗真菌、抗原虫和抗生素类药物。其他知识 关于叶酸前体的生物合成可能有助于设计和 发现同样有用的叶酸抑制剂。Rho-氨基苯甲酸酯是 由分支酸和谷氨酰胺经两步反应合成。Rho- 氨基苯甲酸合成酶(PS)将分支酸和谷氨酰胺转化为 中间体，然后被一种新发现的酶转化为PABA 仅被称为“酶X”。PS是一种二聚体酶，由脱链的 基因、PABA和PAB。PABA和PAB都已测序，并正在 与另外两种使用分支酸的酶同源，邻氨基苯甲酸酯 合酶和异分支酸合酶。为了更好地理解 这些酶的进化、结构和代谢关系， 这项提议试图描述罗氨基苯甲酸酯的酶的特性。 合成，以及编码它们的基因的表达。身份 Rho-氨基苯甲酸酯中中间体的含量将通过分离 从酶反应中得到化合物，然后进行核磁共振分析。这些酶 将被提纯到均质，它们的动力学参数将是 下定决心。酶活性的小分子效应器也将 调查过了。PABA和PAB将使用这两种寡核苷酸进行诱变 定向和化学诱变。氨基酸残基在人体内起重要作用 催化活性、蛋白质结构、亚基相互作用和反馈 抵抗力将被确定。每种基因的表达 将对PS和酶X的编码进行研究。这些基因是不相连的， 显然对Rho-氨基苯甲酸或叶酸的反应不受调控。 保持结构性、低水平的机制将由 突变分析。这两种定点突变都使用合成的 寡核苷酸和化学诱变技术将用于 构建和分离影响PABA、pABB和 编码酶X的基因。

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.