A novel carbamoyl-phosphate synthetase from Aquifex aeolicus

Aquifex aeolicus, an extreme hyperthermophile, has neither a full-length ca rbamoyl-phosphate synthetase (CPSase) resembling the enzyme found in all me sophilic organisms nor a carbamate kinase-like CPSase such as those present in several hyperthermophilic archaea. However, the genome has open reading frames encoding putative proteins that are homologous to the major CPSase domains. The glutaminase, CPS.A and CPS.B homologs from A. aeolicus were cl oned, overexpressed in Escherichia coli, and purified to homogeneity. The i solated proteins could catalyze several partial reactions but not the overa ll synthesis of carbamoyl phosphate. However, a stable 124-kDa complex coul d be reconstituted from stoichiometric amounts of CPS.A and CPS.B proteins that synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia. Th e inclusion of the glutaminase subunit resulted in the formation of a 171-k Da complex that could utilize glutamine as the nitrogen-donating substrate, although the catalytic efficiency was significantly compromised. Molecular modeling, using E. coli CPSase as a template, showed that the enzyme has a similar structural organization and interdomain interfaces and that. all o f the residues known to be essential for function are conserved and properl y positioned. A steady state kinetic study at 78 degreesC indicated that al though the substrate affinity was similar for bicarbonate, ammonia, and glu tamine, the K-m for ATP was appreciably higher than that of any known CPSas e. The A. aeolicus complex, with a split gene encoding the major synthetase domains and relatively inefficient coupling of amidotransferase and synthe tase functions, may be more closely related to the ancestral precursor of c ontemporary mesophilic CPSases.