Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

Reduced derivatives of folic acid are required for biosynthesis of DNA, RNA, amino acids, and other important cellular components (14). Folic acid is an essential dietary supplement for humans, while both microorganisms and plants can synthesize this vitamin de novo. The Escherichia coli folic acid biosynthetic pathway is composed of proteins encoded by genes scattered across the chromosome (9); these genes appear to be constitutively expressed at low levels (26, 27). The genes and enzymes involved in folate catabolism in E. coli remain largely unidentified. The abg region of E. coli was first identified in a search for mutants able to grow on folic acid in order to circumvent p-aminobenzoate (PABA) auxotrophy; characterization showed that the auxotrophs were utilizing the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU), not folic acid (Fig. ​(Fig.1)1) (11). The original mutations were point mutations in the intergenic region between abgA and abgR, and it was hypothesized that this resulted in increased expression of the abgABT genes. The abg region, named for enhanced growth on p-aminobenzoyl-glutamate, was mapped to 30 min and was found to consist of a potential operon including abgA, abgB, and abgT. Divergently oriented from abgABT, abgR encodes AbgR, which has homology to LysR-type regulatory proteins (21). Sequence analysis of the putative gene products revealed that AbgA and AbgB were similar to one another and to aminoacyl aminohydrolases and that AbgT was similar to transport proteins (11). FIG. 1. The structures of folic acid, PABA, and PABA-GLU. Previously, we had found that wild-type cells transformed with a high-copy-number plasmid carrying abgT demonstrated saturable uptake of PABA-GLU (KT [transport constant] = 123 μM); control cells harboring the vector alone demonstrated negligible uptake (4). Tritiated PABA-GLU taken in by cells expressing large amounts of AbgT was not rapidly metabolized to a form that was trapped in the cell, as addition of nonradioactive PABA-GLU to these cells resulted in rapid loss of intracellular label. Addition of nonradioactive PABA had no effect. However, experiments with cells harboring complementary plasmids carrying abgT and abgAB, respectively, demonstrated initial uptake of radiolabeled PABA-GLU followed by loss of intracellular label over time. These findings were consistent with the hypothesis that AbgT catalyzed the uptake of PABA-GLU into the cell and that when sufficient amounts of PABA-GLU accumulated, AbgAB catalyzed cleavage to form PABA and glutamate. Since PABA enters cells in a nonsaturable manner consistent with diffusion (27), once the radioactive PABA is generated, it can depart the cell. Taken together, these data supported a model of PABA-GLU utilization in which AbgT catalyzes the import of PABA-GLU, which is subsequently cleaved to PABA by a protein composed of subunits encoded by abgA and abgB. The focus of this study was the purification and characterization of E. coli p-aminobenzoyl-glutamate hydrolase (PGH), encoded by abgA and abgB. This is the first description of an enzyme involved in a potential folate catabolic pathway in E. coli.