K-049. Purification and Characterization of Folate Salvage Enzyme p-Aminobenzoyl-glutamate Lyase from Escherichia coli

J. M. Green, L. Pitstick, E. L. Carter;
Midwestern Univ., Downers Grove, IL.

Background: E. coli contains a cryptic operon that enables utilization of p-aminobenzoyl-glutamate (PABA-GLU), a product of folic acid catabolism. The abg region of the E. coli chromosome includes three genes that encode proteins that enable uptake and growth on the folate breakdown product, p-aminobenzoyl glutamate. Prior studies have demonstrated that AbgT catalyzes import of PABA-GLU, while AbgA and AbgB together cleave PABA-GLU. Hypothesis: We hypothesize that AbgA and AbgB together comprise subunits of an enzyme that cleaves PABA-GLU, and that this enzyme may be purified and characterized. Methods: One-step purification of PABA-GLU hydrolase was accomplished using nickel affinity chromatography of extracts from cells transformed with a high copy plasmid encoding abgAB with a histidine tag. Molecular weights were determined by native and denaturing polyacrylamide gel electrophoresis (PAGE). PABA-GLU hydrolase activity was measured by extracting product PABA into acidified ethyl acetate and measuring either radioactivity or absorbance of p-aminobenzoic acid in the organic layer. Results: Analysis of the protein by sodium dodecyl sulfate PAGE revealed two subunits corresponding to abgB and abgA, in a 2:1 ratio. Native PAGE revealed several bands, with the predominant species corresponding to a molecular weight of ~150 kDa. Excised fragments from an identical, unstained gel were used in an enzyme assay, and the enzyme activity was largely associated with the 150,000 band. Cleavage activity was highly stimulated by the addition of manganese chloride. Kinetic analysis revealed a Km value for PABA-GLU of 80 μM and a turnover number of 90 sec-1.Conclusion: AbgA and AbgB comprise dissimilar subunits of a single manganese-dependent holoenzyme which cleaves PABA-GLU to form PABA and glutamate.
Acknowledgements: This work was supported in part by funds from Midwestern University and grant R15 GM71009 from the National Institutes of Health.