A novel mechanism for resistance to the antimetabolite N-phosphonoacetyl-L-aspartate by Helicobacter pylori.
The mechanism of resistance to N-phosphonoacetyl-L-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes the first committed step of de novo pyrimidine biosynthesis), in Helicobacter pylori was investigated. At a 1 mM concentration, PALA had no effects on the growth and viability of H. pylori. The inhibitor was taken up by H. pylori cells and the transport was saturable, with a Km of 14.8 mM and a Vmax of 19.1 nmol min-1 microliters of cell water-1. By 31P nuclear magnetic resonance (NMR) spectroscopy, both PALA and phosphonoacetate were shown to have been metabolized in all isolates of H. pylori studied. A main metabolic end product was identified as inorganic phosphate, suggesting the presence of an enzyme activity which cleaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate group cleavage was saturable, and there was no evidence for substrate inhibition at higher concentrations of either compound. C-P bond cleavage activity was temperature dependent, and the activity was lost in the presence of the metal chelator EDTA. Other cleavages of PALA were observed by 1H NMR spectroscopy, with succinate and malate released as main products. These metabolic products were also formed when N-acetyl-L-aspartate was incubated with H. pylori lysates, suggesting the action of an aspartase. Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the soluble fraction and that the aspartase activity appeared in the membrane-associated fraction. The results suggested that the two H. pylori enzymes transformed the inhibitor into noncytotoxic products, thus providing the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.
Burns
B P
BP
School of Microbiology and Immunology, University of New South Wales, Sydney 2052, Australia.
Mendz
G L
GL
Hazell
S L
SL
D000963:Antimetabolites / Q000378:metabolism / Q000494:pharmacology*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport; D004352:Drug Resistance, Microbial; D006131:Growth Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D016480:Helicobacter pylori / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D007700:Kinetics; D008670:Metals; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D014316:Tritium
D000963:Antimetabolites; D006131:Growth Inhibitors; D008670:Metals; D014316:Tritium; D001224:Aspartic Acid; C013195:sparfosic acid; D010746:Phosphonoacetic Acid