Abstract
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.
Highlights
Reduced folates and their various one-carbon derivatives are essential for growth and cell division of plants, animals, and bacteria
BLAST searches were performed using the amino acid sequence of both AbgA and AbgB, respectively, and all proteins that were identified as homologous were from bacteria, suggesting that p-aminobenzoyl-glutamate hydrolase (PGH) is only found in prokaryotes
While we theorized that each PGH subunit might perform one of the functions of Ps-carboxypeptidase G2 (CPG2), i.e., subunit interaction or catalytic function, the homology among AbgB, AbgA and Ps-CPG2 spans the entire length of the proteins
Summary
Reduced folates and their various one-carbon derivatives are essential for growth and cell division of plants, animals, and bacteria. Humans cannot synthesize this vitamin and require folate in their diet, in contrast to plants and bacteria which can make folate [1]. This makes the folate biosynthetic pathway an ideal target for antibiotics. The sulfa drugs, which were among the first antibiotics, inhibit the folate biosynthetic pathway. While antibiotic resistance has developed in response to each of these, together these two drugs are still commonly and successfully used in treatment of bacterial infections
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