A Novel Mechanism for Resistance to the Antimetabolite N -Phosphonoacetyl- l -Aspartate by Helicobacter pylori

Abstract

ABSTRACT 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 K m of 14.8 mM and a V max of 19.1 nmol min −1 μl of cell water −1 . By 31 P 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 1 H 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.