The 1.5 angstrom resolution crystal structure of the carbamate kinase-likecarbamoyl phosphate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability incarbamate kinases

Carbamoyl phosphate (CP), an essential precursor of arginine and the pyrimi dine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kina se (CK), an enzyme used by microorganisms to produce Am from ADP and CP. Al though the recently determined structures of CPS and CK show no obvious mut ual similarities, a CK-like CPS reported in hyperthermophilic archaea was p ostulated to be a missing Link in the evolution of CP biosynthesis. The 1.5 Angstrom resolution structure of this enzyme from Pyrococcus furiosus show s both a subunit topology and a homodimeric molecular organization, with a 16-stranded open beta-sheet core surrounded by alpha-helices, similar to th ose in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and pr esents a bound,ADP molecule, which does not dissociate at 22 degrees C from the enzyme. The ADP nucleotide is sequestered in a ridge formed over the C -edge of the core sheet, at the bottom of a large cavity, with the purine r ing enclosed in a pocket specific for adenine. Overall, the enzyme structur e is ill-suited for catalyzing the characteristic three-step reaction of CP S and supports the view that the CK-Like CPS is in fact a highly thermostab le and very slow (at 37 degrees C) CK that, in the extreme environment of P . furiosus, may have the new function of making, rather than using, CP. The thermostability of the enzyme may result from the extension of the hydroph obic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair net-works across several secondary structure el ements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests thar: the slow rat e at 37 degrees C is possibly a consequence of slow product dissociation. (C) 2000 Academic Press.