ENZYMES AND GENES OF P-AMINOBENZOATE BIOSYNTHESIS

对氨基苯甲酸酯（PABA）是一种芳香族化合物， 是它与维生素二氢叶酸的结合。 的衍生物 二氢叶酸参与细胞的“一碳”代谢， 参与DNA、蛋白质和脂质前体的生物合成。 因为叶酸是大分子代谢的中心， 生物合成途径已经成为几种生物学方法的目标， 抗真菌剂、抗原虫剂和抗生素剂。 额外知识 关于叶酸前体的生物合成可能有助于设计和 发现类似有用的叶酸抑制剂。 对氨基苯甲酸酯是 由分支酸和谷氨酰胺以两步途径合成。 ρ- 氨基苯甲酸合酶（PS）将分支酸和谷氨酰胺转化为氨基苯甲酸盐。 中间体，然后通过新发现的酶转化为PABA 被称为“X酶” PS是一种二聚体酶， pabA和pabB基因。 pabA和pabB都已测序， 与其他两种使用分支酸盐、邻氨基苯甲酸盐的酶同源 合酶和异分支酸合酶。 为了解 这些酶的进化、结构和代谢关系， 该建议试图表征对-氨基苯甲酸酯的酶 合成，以及编码它们的基因的表达。 身份 将通过分离 该化合物来自酶反应，随后进行NMR分析。 的酶 将被纯化至均匀，并且它们的动力学参数将是 测定 酶活性的小分子效应物也将是 研究了 将使用两种寡核苷酸对pabA和pabB进行诱变 定向诱变和化学诱变。 重要的氨基酸残基 催化活性、蛋白质结构、亚基相互作用和反馈 阻力将被发现。 每个基因的表达 将研究编码PS和酶X的基因。 基因是不相连的， 对邻氨基苯甲酸盐或叶酸盐的反应明显不受调节。 维持组成性低水平的机制将通过以下方式进行研究： 突变分析 两种定点诱变都使用合成的 寡核苷酸和化学诱变技术将用于 构建并分离影响pabA、pabB和pabC表达的突变， 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.