THE CARBAMATE KINASE-LIKE CARBAMOYL-PHOSPHATE SYNTHETASE OF THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS, A MISSING LINK IN THE EVOLUTION OF CARBAMOYL-PHOSPHATE BIOSYNTHESIS

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitro gen donor and are composed of two subunits (or domains), one exhibitin g glutaminase activity, the other able to synthesize carbamoyl phospha te (CP) from bicarbonate, ATP, and ammonia, The pseudodimeric organiza tion of this synthetase suggested that it has evolved by duplication o f a smaller kinase, possibly a carbamate kinase (CK), In contrast to o ther prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus wa s found to synthesize CP by using ammonia and not glutamine, We have p urified the cognate enzyme and found it to be a dimer of two identical subunits of M-r 32,000. Its thermostability is considerable, 50% acti vity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C, The corresponding gene was cloned by PCR and found to present about 5 0% amino acid identity with known CKs, The stoichiometry of the reacti on (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction howe ver clearly distinguish P. furiosus CPS from ordinary CKs, Thus the CP S of P. furiosus could represent a primeval step in the evolution of C PS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have prece ded the duplication assumed to have generated the two subdomains of mo dern CPSs, The gene coding for this CK-like CPS was called cpkA.