The Carbamate Kinase-Like Carbamoyl Phosphate Synthetase of The

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12803–12808, November 1997 Biochemistry The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis VIRGINIE DURBECQ*, CHRISTIANNE LEGRAIN†,MARTINE ROOVERS‡§,ANDRE´ PIE´RARD*†, AND NICOLAS GLANSDORFF†‡§¶ *Laboratoire de Microbiologie, Universite´Libre de Bruxelles, †Research Institute of the Centre de Recherches et d’Enseignement des Industries Alimentaires et Chimiques, ‡Microbiologie, Vrije Universiteit Brussel, and §Flanders Interuniversity Institute for Biotechnology, Avenue E. Gryson 1, B-1070 Brussels, Belgium Communicated by H. Edwin Umbarger, Purdue University, West Lafayette, IN, September 16, 1997 (received for review July 9, 1997) ABSTRACT Microbial carbamoyl phosphate synthetases protein, and it was proposed that the synthetase gene had (CPS) use glutamine as nitrogen donor and are composed of evolved by duplication of a smaller ancestral gene—possibly two subunits (or domains), one exhibiting glutaminase activ- coding for a carbamate kinase (CK) (5). CKs (EC 2.7.2.2) are ity, the other able to synthesize carbamoyl phosphate (CP) homodimers composed of subunits of 31–37 kDa (6). As from bicarbonate, ATP, and ammonia. The pseudodimeric carbamate is in chemical equilibrium with bicarbonate and 2 organization of this synthetase suggested that it has evolved by ammonia, CK can synthesize CP from HCO3 ,NH3 and one duplication of a smaller kinase, possibly a carbamate kinase molecule of ATP (Fig. 1). This evolutionary scheme found (CK). In contrast to other prokaryotes the hyperthermophilic support in the weak but nevertheless suggestive sequence archaeon Pyrococcus furiosus was found to synthesize CP by similarities that were brought to light between carB and the using ammonia and not glutamine. We have purified the gene for CK in Pseudomonas aeruginosa (7). Moreover, further cognate enzyme and found it to be a dimer of two identical studies showed that the N-terminal half of the synthetase subunits of Mr 32,000. Its thermostability is considerable, 50% (subdomain CPS.A) catalyzed the synthesis of carboxyphos- activity being retained after1hat100°C or3hat95°C. The phate (Fig. 1, reaction 1), whereas the C-terminal part was corresponding gene was cloned by PCR and found to present responsible for the second ATP-dependent reaction, the syn- about 50% amino acid identity with known CKs. The stoichi- thesis of CP (Fig. 1, reaction 3) (8–11). This evidence supports ometry of the reaction (two ATP consumed per CP synthe- the suggestion that each one of the copies of the ancestral sized) and the ability of the enzyme to catalyze at high rate a kinase has become specialized in one of the two ATP- bicarbonate-dependent ATPase reaction however clearly dis- dependent reactions involved in CP synthesis (12–14). tinguish P. furiosus CPS from ordinary CKs. Thus the CPS of We have reported a thermostable CPS activity in the P. furiosus could represent a primeval step in the evolution of hyperthermophile archaeon Pyrococcus furiosus. This activity CPS from CK. Our results suggest that the first event in this appears unique among the microorganisms investigated so far evolution was the emergence of a primeval synthetase com- in that ammonia and not glutamine is the nitrogen donor of the posed of subunits able to synthesize both carboxyphosphate reaction (15). The enzyme appears to channel the very ther- and CP; this step would have preceded the duplication as- molabile CP molecule toward citrulline, presumably by asso- sumed to have generated the two subdomains of modern CPSs. ciating with ornithine transcarbamylase (OTC) in a multien- The gene coding for this CK-like CPS was called cpkA. zyme complex (15). In the present study, the enzyme was purified 350-fold and the protein studied in detail. The corresponding gene was cloned and its sequence determined. Carbamoyl phosphate (CP) is a key metabolite in nitrogen Whereas the genetic data reveal substantial similarities with metabolism because it is a precursor of both arginine and the CKs, several properties of the enzyme—such as the stoichi- pyrimidines. Three classes of carbamoyl phosphate synthetases ometry of the reaction and its ability to catalyze a high-rate, (CPS, EC 6.3.5.5) have been recognized on the basis of their bicarbonate-dependent ATPase reaction—clearly distinguish substrate specificity—whether glutamine or ammonia is used it from ordinary CKs. as the nitrogen donor—and their requirement for N-acetyl-L- The data support the hypothesis that P. furiosus CPS indeed glutamate as allosteric effector (reviewed in refs. 1 and 2). is related to the ancestral form of the synthetase component of Despite this apparent diversity, all three forms of CPS consist CPSs; however, they also suggest that the first step in the of two entities, a glutaminase domain (or subunit) and a evolution from an ancestral CK toward CPS consisted of synthetase domain (or subunit) that catalyzes ATP-dependent mutations conferring to the CK subunit the capacity to cata- synthesis of CP from ammonia and bicarbonate. In prokaryotic lyze either one of the two ATP-dependent reactions. and fungal arginine-specific CPSs the two functions are ful- filled by separate polypeptides, whereas in other organisms the homologous parts of the molecule are domains within the same MATERIALS AND METHODS polypeptide. The synthesis of CP by the synthetase domain or 9 Chemicals. P1.P3 di-(adenosine-5 )-triphosphate (Ap3A), subunit of CPS proceeds in three consecutive steps (1, 3, 4); 9 a b P1.P5 di-(adenosine-5 )-pentaphosphate (Ap5A), and , two ATP molecules are consumed in the process (Fig. 1). Analysis of the DNA sequence encoding the synthetase Abbreviations: CPS, carbamoyl phosphate synthetase; CK, carbamate subunit of Escherichia coli (the carB gene) revealed consider- kinase; CP, carbamoyl phosphate; OTC, ornithine transcarbamylase; able similarity between the N- and C-terminal halves of the Ap3A, P1.P3 di-(adenosine-59)-triphosphate; Ap5A, P1.P5 di- (adenosine-59)-pentaphosphate. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. Y09829). payment. This article must therefore be hereby marked ‘‘advertisement’’ in ¶To whom reprint requests should be addressed at: Centre de Re- accordance with 18 U.S.C. §1734 solely to indicate this fact. cherches et d’Enseignement des Industries Alimentaires et © 1997 by The National Academy of Sciences 0027-8424y97y9412803-6$2.00y0 Chimiques, Institut de Recherches, Avenue E. Gryson 1, B-1070 PNAS is available online at http:yywww.pnas.org. Brussels, Belgium. e-mail: [email protected]. 12803 Downloaded by guest on September 30, 2021 12804 Biochemistry: Durbecq et al. Proc. Natl. Acad. Sci. USA 94 (1997) Sepharose CL6B 26y40 column equilibrated with 50 mM TriszHCl buffer (pH 7.2). The CPS activity was eluted with a linear gradient of 0–0.5 M KCl in 50 mM TriszHCl buffer (pH 7.2). The active fractions were pooled, concentrated, dialyzed against 50 mM TriszHCl buffer (pH 7.2), and applied on a Blue Sepharose 16y16 column equilibrated with 20 mM TriszHCl buffer (pH 7.2) and 5 mM MgCl2. The column was washed with z 20 mM Tris HCl buffer (pH 7.2) and 5 mM MgCl2. The CPS activity was eluted with 50 mM TriszHCl buffer (pH 7.2) and 20 mM MgATP. The active fractions were collected, concentrated and applied on a Sephadex G200 26y40 column equilibrated with 50 mM TriszHCl buffer (pH 7.2). Active fractions were collected and applied to a Superose P12 HR 10y30 (fast protein liquid chromatography, FPLC) equilibrated with 50 z FIG. 1. Reactions catalyzed by CPS (1–3), by CK (4), and chemical mM Tris HCl buffer (pH 7.2). For the last step, active fractions equilibrium of carbamate with bicarbonate and ammonia (5). were applied to a Mono-Q HR 5y5 column (FPLC) equilibrated with 50 mM TriszHCl buffer (pH 7.2) and eluted with 14 methylene-ATP were from Sigma. Na2 CO3 was from Am- a linear gradient of KCl 0–0.5 M in 50 mM TriszHCl. ersham. The Original TA Cloning Kit was from Invitrogen. Electrophoresis. Electrophoresis under native and denatur- Purified CPS of E. coli was from this laboratory (courtesy of ing conditions (SDS) was performed on 8–25% gradient gels S. Delannay). in the PHAST System (Pharmacia). Protein bands were visu- Bacterial Strain and Culture Conditions. P. furiosus strain alized by staining with Coomassie brilliant blue. Vc1 (DSM 3638) (16) was grown at 95°C under anaerobic Cloning of the CK-Like CPS Gene. Cloning was achieved by conditions as described (15). Streptococcus faecalis strain PCR with Taq polymerase (15 sec at 94°C, 60 sec at 48°C, 30 ATCC 11700 was grown on complex medium with 0.5% sec at 72°C, 30 cycles) on 1 mg of genomic DNA of P. furiosus arginine and 2.5% glucose. restricted by PstI and primed with 50 pmol of two degenerate CPS Assay. Activity was measured as previously described oligonucleotides (GIBCOyBRL): 59-CAY GGN AAY GGN (15) except that MgATP concentration was 3 mM instead of CCH CA-39 and 59-TCY TTR TCD ATN ACD GC-39 [n 5 A 10 mM. One enzyme unit is defined as the amount of enzyme 1 1 1 5 1 5 1 5 1 1 5 2 C T G, r A G, Y C T, H A T C, D that converts 1 mmol substrate to product h 1. A 1 T 1 G] corresponding to two conserved regions of CK Determination of Protein. Protein was measured by the genes (see Fig. 3). A first fragment of 501 bp related to the Lowry method (17), or in the last purification steps by UV middle of the gene was cloned into the TA cloning kit vector.

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