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

Abstract

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (ϳ53-kDa and ϳ47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K m value for PABA-GLU of 60 ؎ 0.08 M and a specific activity of 63,300 ؎ 600 nmol min ؊1 mg ؊1 . Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.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 paminobenzoate (PABA) auxotrophy; characterization showed that the auxotrophs were utilizing the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU), not folic acid ( Fig. 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).Previously, we had found that wild-typ...

Comparison of Substrate Specificity of <em>Escherichia Coli</em> p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em> Carboxypeptidase G

“…SDS-PAGE gels (7.5%) were obtained from Bio-Rad (Hercules, CA). E. coli PGH was isolated from E. coli as previously described [15]. …”

“…The assay for cleavage of PABA-GLU was performed essentially as described previously [15]. Reaction mixtures consisted of 50 mM Tris, pH 8.5, 10 mM β -mercaptoethanol, 5 mM MnCl 2 , and varying concentrations of PABA-GLU.…”

“…Samples (0.5 mL) were taken with time (30, 60, 90 and 120 sec) and terminated with addition of 1.0 M sodium citrate, pH 5.5 (0.5 mL). Product p -aminobenzoate (PABA) was extracted into 2 mL of ethyl acetate, and absorbance of the ethyl acetate layer containing PABA was measured at 284 nm ( ε = 13,400 M −1 ∙cm −1 [15]). The velocity was calculated as the slope of the line (nmoles of product versus time in minutes).…”

“…Enzyme reaction mixtures were analyzed for glutamate content using an HPLC-based method, as described previously [15]. The procedure involved removal of PGH or CPG with a filter device, followed by modification of an aliquot of reaction mixture by dabsylation.…”

“…Very little was known about gene organization and folate catabolism in bacteria until the abg region was identified in E. coli [13]. AbgT imports PABA-GLU [14], while abgA and abgB encode subunits of the heterotetrameric p- aminobenzoyl-glutamate hydrolase (PGH) [15]. This study also demonstrated that PGH required divalent manganese for activity, and failed to cleave a range of dipeptides.…”