PMID:20190044
| Citation |
Green, JM, Hollandsworth, R, Pitstick, L and Carter, EL (2010) Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli. J. Bacteriol. 192:2407-13 |
|---|---|
| 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 (approximately 53-kDa and approximately 47-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 microM 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. |
| Links |
PubMed PMC2863484 Online version:10.1128/JB.01362-09 |
| Keywords |
4-Aminobenzoic Acid/chemistry; 4-Aminobenzoic Acid/metabolism; Chromatography, Affinity; Chromatography, Gel; Escherichia coli/enzymology; Escherichia coli/isolation & purification; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Folic Acid/chemistry; Folic Acid/metabolism; Hydrolases/isolation & purification; Hydrolases/metabolism; Kinetics; Molecular Structure; Polymerase Chain Reaction; gamma-Glutamyl Hydrolase/isolation & purification; gamma-Glutamyl Hydrolase/metabolism |
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