Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min ؊1 mg of protein ؊1 and had an apparent K m for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min ؊1 mg of protein
؊1. Hypoxanthine PRTase activity was present at 0.85 nmol min ؊1 mg of protein ؊1 with an apparent K m for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min ؊1 mg of protein
؊1. PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.De novo synthesis of purine nucleotides requires a minimum of 10 enzymes for the production of IMP, the major intermediate of purine metabolism (6). The salvage of nucleobases and nucleosides provides an energy-saving alternative. Salvage pathways have been identified in eubacteria, eucaryotes, and archaebacteria and comprise a collection of enzymes capable of converting preformed nucleobases and nucleosides to AMP, GMP, IMP, and XMP (5,7,10,13,17,19,21,23,29). These enzymes enable organisms to use exogenous bases liberated from lysed cells or recycle those produced by the breakdown of unstable RNA. A key enzyme in the pathway is phosphoribosyltransferase (PRTase). This enzyme is responsible for the conversion of bases to nucleotide 5Ј-monophosphates by use of 5-phosphorylribose-1-pyrophosphate (PPRP) as the phosphopentose donor. Bases can also be salvaged by nucleoside phosphorylases by use of ribose-1-phosphate as the phosphopentose donor. However, in Escherichia coli and Salmonella typhimurium, this mechanism is a minor source of nucleotides (20).Mutants resistant to nucleobase analogs have been isolated from several archaebacteria (2,12,16,29). As with eubacteria and eucaryotes, these mutants have lost the ability to incorporate certain exogenous bases, and this loss is almost always accompanied by a loss of the corresponding PRTase ac...