Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/μg). Relative to its activity with 10 μM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.
Green, J.M. and Matthews, R. G. (2007) Folate Biosynthesis, Reduction, Polyglutamylation and the Interconversion of Folate Derviatives. In: Bock, A., Curtiss III, R., Kaper, J.B., Neidhardt, F.C., Nystrom, T., Rudd, K.E., and Squires, C.L., Eds., EcoSal—Escherichia coli and Salmonella: Cellular and Molecular Biology, Chapter 184.108.40.206, ASM Press, Washington DC.
Albrecht, A.M., Boldizsar, E. and Hutchison, D.J. (1978) Carboxypeptidase Displaying Differential Velocity in Hydrolysis of Methotrexate, 5-Methyltetrahydrofolic Acid, and Leucovorin. Journal of Bacteriology, 134, 506-513.
Green, J.M., Hollandsworth, R., Pitstick, L. and Carter, E.L. (2010) Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli. Journal of Bacteriology, 192, 2407-2413. http://dx.doi.org/10.1128/JB.01362-09
Rowsell, S., Pauptit, R.A., Tucker, A.D., Melton, R.G., Blow, D.M. and Brick, P. (1997) Crystal Structure of Carboxypeptidase G2, a Bacterial Enzyme with Applications in Cancer Therapy. Structure, 5, 337-347.
Sherwood, R.F., Melton, R.G., Alwan, S. M. and Hughes, P. (1985) Purification and Properties of Carboxypeptidase G2 from Pseudomonas sp. Strain RS-16. Use of a Novel Triazine Dye Affinity Method. European Journal of Biochemistry, 148, 447-453. http://dx.doi.org/10.1111/j.1432-1033.1985.tb08860.x
Minton, N.P., Atkinson, T. and Sherwood, R.F. (1983) Molecular Cloning of the Pseudomonas Carboxypeptidase G2 Gene and Its Expression in Escherichia coli and Pseudomonas putida. Journal of Bacteriology, 156, 1222-1227.
Widemann, B.C., Balis, F.M., Kim, A., Boron, M., Jayaprakash, N., Shalabi, A., O’Brien, M., Eby, M., Cole, D.E., Murphy, R.F., Fox, E., Ivy, P. and Adamson, P.C. (2010) Glucarpidase, Leucovorin, and Thymidine for High-Dose Methotrexate-Induced Renal Dysfunction: Clinical and Pharmacologic Factors Affecting Outcome. Journal of Clinical Oncology, 28, 3979-3986. http://dx.doi.org/10.1200/JCO.2009.25.4540
Wood, R.C. and Hitchings, G.H. (1959) A Study of the Uptake and Degradation of Folic Acid, Citrovorum Factor, Aminopterin, and Pyrimethamine by Bacteria. Journal of Biological Chemistry, 234, 2381-2385.
Suh, J.R., Oppenheim, E.W., Girgis, S. and Stover, P.J. (2000) Purification and Properties of a Folate-Catabolizing Enzyme. Journal of Biological Chemistry, 275, 35646-35655. http://dx.doi.org/10.1074/jbc.M005864200
Richey, D.P. and Brown, G.M. (1970) A Comparison of the Effectiveness with Which p-Aminobenzoic Acid and p-Aminobenzoylglutamic Acid Are Used as Substrate by Dihydropteroate Synthetase from Escherichia coli. Biochimica et Biophysica Acta (BBA)—General Subjects, 222, 237-239. http://dx.doi.org/10.1016/0304-4165(70)90376-4