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