Ammonia-dependent synthesis and metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus

1 Research Institute, CERIA-COOVl, Vrije Universiteit Brussel, Brussels, Belgium 2 Laboratorium voor Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Brussels, Belgium 3 Vlaams Interuniversitair Instituut voor Biotechnologie, Université Libre de Bruxelles, 1, avenue Emile Gryson, B-1070 Brussels, Belgium 4 Laboratoire de Microbiologie, Université Libre de Bruxelles, 1, avenue Emile Gryson, B-1070 Brussels, Belgium 5 Tel: +32 2 5267276. Fax: +32 2 5267273. e-mail: ceriair@ulb.ac.be ABSTRACT The biosynthesis of carbamoyl phosphate (CP), a metabolic precursor of arginine and the pyrimidines was investigated in the hyperthermophilic archaeon Pyrococcus furiosus . The half-life of CP was found to be less than 2 s in the optimum temperature range of this organism (100-102 °C). The carbamoyl-phosphate synthase (CPSase) of P. furiosus uses ammonia as the nitrogen donor, and not glutamine like all micro-organisms investigated so far. The Mr of the enzyme, which is devoid of regulatory properties, is 70000, at variance with that of known CPSases. The possible significance of these findings with regard to hyperthermophilic nitrogen metabolism is discussed. Competition experiments with P. furiosus crude extracts indicated a marked preference of ornithine carbamoyltransferase (OTCase) for CP synthesized by CPSase rather than for CP added to the reaction mixture. In addition, the bisubstrate analogue - N -phosphonoacetyl-L-ornithine inhibits the formation of citrulline from bicarbonate, ammonia, ATP and ornithine much less than its synthesis from ornithine and CP in the presence of free OTCase. Such results suggest that, in vivo , CPSase and OTCase associate in a complex able to channel CP. Such a channelling may confer protection to CP, thus avoiding the accumulation of toxic amounts of cyanate arising from its decomposition as well as the waste of the two molecules of ATP required for its synthesis. Keywords: Pyrococcus furiosus, ammonia-dependent carbamoyl-phosphate synthase, thermal lability of cirbamoyl phosphate, metabolic channelling