Carbamoylphosphate Metabolism in Thermophilic Archaea
Virginie DURBECQ*a, Christianne LEGRAINb, Martine ROOVERSc,a, Thia-Lin THIA-TOONGc,d, Daniel CHARLIERb,d, Andre PIERARDa, b, and Nicolas GLANSDORFFb,c,d
a Laboratoire de Microbiologie, Universite Libre de Bruxelles, Ave. E. Gryson 1, B-1070 Brussels, Belgium
b Research Institute of the CERIA-COOVI, Ave. E. Gryson 1, B-1070 Brussels, Belgium
c Microbiologie, Vrije Universiteit Brussel, Ave. E. Gryson 1, B- 1070 Brussels, Belgium
d Flanders Interuniversity Institute for Biotechnology, Ave. E. Gryson 1, B-1070 Brussels, Belgium
Carbamoylphosphate (CP) is an essential metabolite for both arginine and pyrimidine biosynthetic pathways; ornithine carbamoyltransferase (OTCase) catalyzes the formation of citrulline from ornithine and CP in the sixth step of arginine biosynthesis, whereas aspartate carbamoyltransferase (ATCase) promotes the condensation of aspartate and CP into carbamoylaspartate in the first committed step of pyrimidine biosynthesis. CP is synthesized in vivo by a carbamoylphosphate synthetase (CPSase); it is a particularly thermolabile compound and its thermal decomposition yields cyanate, a powerful carbamoylating and therefore toxic agent (1, 2). The metabolism of CP was investigated in the hyper- and extremely thermophilic archaea Pyrococcus furiosus and Sulfolobus solfataricus.
P. furiosus CPSase was purified and found to be a dimer of two identical subunits of Mr 32,000 showing considerable thermostability. The corresponding gene presents about 50% amino acid identity with known carbamate kinases (CKases). The stoichiometry of the reaction and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P furiosus CPSase from CKases. Thus the CPSase of P furiosus could represent a primeval step in the evolution of CPSase from CK (3). P furiosus OTCase (4) and CPSase were expressed in S. cerevisiae. This property offers the prospect of analysing the specificity of interaction between the two proteins by the yeast 2-hybrid system.
The S. solfataricus pyrBI genes encoding ATCase were cloned by complementation. They are clustered into an Enterobacterial-like pyrBI operon and are transcribed from a typical archaeal promoter. The Sulfolobus pyrBI operon is part of a larger pyrimidine gene cluster, comprising also pyrE, C and D, in which pyrE and pyrBI constitute the wings of a divergent operon. The deduced pyrB and pyrI amino acid sequences bear 48 to 64% similarity to archaeal and eubacterial ATCases. The pyrB gene and the pyrBI operon were subcloned in E. coli and the corresponding products characterised. The pyrBI encoded holoenzyme is highly thermostable
