Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli.
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.
Green
Jacalyn M
JM
Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. jgreen@midwestern.edu
Hollandsworth
Ryan
R
Pitstick
Lenore
L
Carter
Eric L
EL
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*
D029968:Escherichia coli Proteins; D005492:Folic Acid; D006867:Hydrolases; D011623:gamma-Glutamyl Hydrolase; C549014:p-aminobenzoyl-glutamate hydrolase, E coli; D010129:4-Aminobenzoic Acid