The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

Abstract

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity 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 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS 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 preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.

Figure 1

Reactions catalyzed by CPS (–3), by CK (4), and chemical equilibrium of carbamate with bicarbonate and ammonia (5).

Figure 3

Nucleotides and deduced amino acid sequence of the P. furiosus CPS. Numbering begins at the first nucleotide of the sequence. The A-box motif is indicated by stars (✽), B-box motif by circle (○), and termination site by double points (:). Upstream and downstream primers used for degenerated PCR are underlined; upstream and downstream primers used for inverse PCR are overlined. Amino acid sequence determined by Edman degradation is flanked by two triangles.

Figure 4

Multiple alignment of amino acid sequences (species code and GenBank accession numbers in parentheses) of the CPS from P. furiosus (Pyrfu; Y09829) and CK from Pseudomonas aeruginosa (Pseae; X14693), Clostridium perfringens (Clope; X97768), Halobacterium salinarium (Halsa; X80931), Synechocystis sp (Synec; D90917), and Hemophilus influenzae (Hemin; U32741). Identical residues are marked with asterisks, and conservative substitutions are marked with points. Amino acid sequence determined by Edman degradation is flanked by two triangles.

Figure 2

Stoichiometry of ATP consumption and CP production in the reaction catalyzed by purified P. furiosus CPS (A) and S. faecalis CK (B).

Figure 5

Scheme illustrating the hypothesis presented in this paper for the evolution of the modern CPS synthetase subunit from a dimeric CK (K). The nitrogen donor (NH₃) is not represented nor the glutaminase subunit which, in a later step, is assumed to have combined with the synthetase subunit, conferring upon the enzyme the ability to use glutamine as nitrogen donor. During the transition between steps II and III, fusion (or recombination) joining the primeval synthetase gene (K′) may have preceded divergent evolution of the two copies of the primeval synthetase gene (K′) toward subdomains CPS.A and CPS.B.