A NOVEL MECHANISM FOR RESISTANCE TO THE ANTIMETABOLITE N-PHOSPHONOACETYL-L-ASPARTATE BY HELICOBACTER-PYLORI

The mechanism of resistance to N-phosphunoacetyl-L-aspartate (PALA), a potent inhibitor of aspartate carbamoyltransferase (which catalyzes t he first committed step of de novo pyrimidine biosynthesis), in Helico bacter pylori was investigated, At a 1 mM concentration, PALA had no e ffects on the grow-th and viability of H. pylori. The inhibitor was ta ken up by H. pylori cells and the transport was saturable, with a K-m of 14.8 mM and a V-max of 19.1 nmol min(-1) mu l of cell water(-1), By P-31 nuclear magnetic resonance (NMR) spectroscopy, both PALA and pho sphonoacetate were shown to have been metabolized in all isolates of H . pylori studied. A main metabolic end product was identified as inorg anic phosphate, suggesting the presence of an enzyme activity which cl eaved the carbon-phosphorus (C-P) bonds. The kinetics of phosphonate g roup cleavage,vas saturable, and there was no evidence for substrate i nhibition at higher concentrations of either compound. C-P bond cleava ge activity was temperature dependent, and the activity was lost in th e presence of the metal chelator EDTA, Other cleavages of PALA were ob served by H-1 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 acti on of an aspartase, Studies of the cellular location of these enzymes revealed that the C-P bond cleavage activity was localized in the solu ble fraction and that the aspartase activity appeared in the membrane- associated fraction. The results suggested that the two H. pylori enzy mes transformed the inhibitor into noncytotoxic products, thus providi ng the bacterium with a mechanism of resistance to PALA toxicity which appears to be unique.