title: 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 (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.
authors.affiliation: Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. jgreen@midwestern.edu
authors.forename: Jacalyn M
authors.initials: JM
authors.lastname: Green
authors.forename: Ryan
authors.initials: R
authors.lastname: Hollandsworth
authors.forename: Lenore
authors.initials: L
authors.lastname: Pitstick
authors.forename: Eric L
authors.initials: EL
authors.lastname: Carter
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism*; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D011623:gamma-Glutamyl Hydrolase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D005492:Folic Acid; D006867:Hydrolases; D011623:gamma-Glutamyl Hydrolase; C549014:p-aminobenzoyl-glutamate hydrolase, E coli; D010129:4-Aminobenzoic Acid