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.
