Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli. 2010

Jacalyn M Green, and Ryan Hollandsworth, and Lenore Pitstick, and Eric L Carter
Department of Biochemistry, Midwestern University, 555 31st Street, Downers Grove, IL 60515, USA. jgreen@midwestern.edu

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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D010129 4-Aminobenzoic Acid An aminobenzoic acid isomer that combines with pteridine and GLUTAMIC ACID to form FOLIC ACID. The fact that 4-aminobenzoic acid absorbs light throughout the UVB range has also resulted in its use as an ingredient in SUNSCREENS. PABA,p-Aminobenzoic Acid,para-Aminobenzoic Acid,4-Aminobenzoic Acid, Potassium Salt,Aminobenzoic Acid (USP),Epit Vit,Epitelplast,Hachemina,Magnesium para-Aminobenzoate,Pabasan,Paraminan,Paraminol,Potaba,Potassium 4-Aminobenzoate,Potassium Aminobenzoate,4 Aminobenzoic Acid,4 Aminobenzoic Acid, Potassium Salt,4-Aminobenzoate, Potassium,Aminobenzoate, Potassium,Potassium 4 Aminobenzoate,p Aminobenzoic Acid,para Aminobenzoic Acid,para-Aminobenzoate, Magnesium
D011623 gamma-Glutamyl Hydrolase Catalyzes the hydrolysis of pteroylpolyglutamic acids in gamma linkage to pterolylmonoglutamic acid and free glutamic acid. EC 3.4.19.9. Conjugase,Folate Conjugase,Folyl Conjugate Synthetase,Pteroyl Polyglutamate Hydrolase,Carboxypeptidase G,Carboxypeptidase G1,Carboxypeptidase G2,Folacin Conjugase,Folate Hydrolyzing Enzyme,Folyl Poly-gamma-Glutamate Carboxypeptidase,Folyl Polyglutamate Cleavage Enzyme,Folylpolyglutamate Hydrolase,gamma Glutamyl Hydrolase
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005492 Folic Acid A member of the vitamin B family that stimulates the hematopoietic system. It is present in the liver and kidney and is found in mushrooms, spinach, yeast, green leaves, and grasses (POACEAE). Folic acid is used in the treatment and prevention of folate deficiencies and megaloblastic anemia. Pteroylglutamic Acid,Vitamin M,Folacin,Folate,Folic Acid, (D)-Isomer,Folic Acid, (DL)-Isomer,Folic Acid, Calcium Salt (1:1),Folic Acid, Monopotassium Salt,Folic Acid, Monosodium Salt,Folic Acid, Potassium Salt,Folic Acid, Sodium Salt,Folvite,Vitamin B9,B9, Vitamin
D006867 Hydrolases Any member of the class of enzymes that catalyze the cleavage of the substrate and the addition of water to the resulting molecules, e.g., ESTERASES, glycosidases (GLYCOSIDE HYDROLASES), lipases, NUCLEOTIDASES, peptidases (PEPTIDE HYDROLASES), and phosphatases (PHOSPHORIC MONOESTER HYDROLASES). EC 3. Hydrolase
D015394 Molecular Structure The location of the atoms, groups or ions relative to one another in a molecule, as well as the number, type and location of covalent bonds. Structure, Molecular,Molecular Structures,Structures, Molecular
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain

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