Comparison of Substrate Specificity of <em>Escherichia Coli</em> p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em> Carboxypeptidase G

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.

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague–Dawley Rats

“…16 In addition, bacterial enzymes such as p -aminobenzoyl-glutamate hydrolase, found in E. coli , have also been shown to catalyze this reaction. 17 As these produced metabolites do not have activity toward DHFR inhibition, glutamate hydrolysis by CPDG2 is seen as a detoxication process. 18 , 19 As such, CPDG2 is approved as a rescue agent in cancer patients presenting delayed MTX clearance and acute nephrotoxicity, 2 and several clinical studies have reported positive results for its use in this role.…”

“…Here, they can undergo further biotransformation by the gut microbiota. , A bacterial enzyme, carboxypeptidase glutamate 2 (CPDG2; also referred to as folylpolyglutamate carboxypeptidase [FGCP]), can cleave the terminal glutamate residues of MTX and 7-OH-MTX , resulting in metabolites such as 2,4-diamino- N -10-methylpteroic acid (DAMPA) and 7-hydroxy-DAMPA, respectively . In addition, bacterial enzymes such as p -aminobenzoyl-glutamate hydrolase, found in E. coli , have also been shown to catalyze this reaction . As these produced metabolites do not have activity toward DHFR inhibition, glutamate hydrolysis by CPDG2 is seen as a detoxication process. , As such, CPDG2 is approved as a rescue agent in cancer patients presenting delayed MTX clearance and acute nephrotoxicity, and several clinical studies have reported positive results for its use in this role. ,, Even so, MTX and its relatively insoluble metabolites are still presumed to precipitate in kidneys, causing nephrotoxicity, as well as causing severe gastrointestinal toxicity such as vomiting, diarrhea, oral mucositis and, in some cases, the death of patients …”

Current evidence and clinical relevance of drug-microbiota interactions in inflammatory bowel disease

“…In addition, bacteria can convert MTX-PG into the inactive metabolite 2,4-diamino-N-10-methylpteroic acid (DAMPA) (Levy and Goldman, 1967;Letertre et al, 2020). Examples of bacterial enzymes catalyzing this latter reaction are the E. coli p-aminobenzoyl-glutamate hydrolase (PGH) and the Pseudomonas carboxypeptidase glutamate 2 (CPDG2) (Larimer et al, 2014). Today, CPDG2 administration is recommended for patients with MTX-PG intoxications (Buchen et al, 2005;Ramsey et al, 2018).…”

Pharmacomicrobiology of Methotrexate in Rheumatoid Arthritis: Gut Microbiome as Predictor of Therapeutic Response

“…The terminal glutamate residues of MTX and 7-OH-MTX can be cleavaged to generate 2, 4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-DAMPA metabolites with the assistance of CPDG2, respectively (41,46,47). In addition, other bacterial enzymes such as p-aminobenzoylglutamate hydrolase, found in E. coli, have also been shown to catalyze this reaction (48) (see Figure 2). The hydrolysis of glutamate by CPDG2 is considered to be a detoxification process.…”