title: Cloning, expression, and structure analysis of carbamate kinase-like carbamoyl phosphate synthetase from Pyrococcus abyssi.
abstract: Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100 degrees C. Carbamoyl phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form carbamoyl phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
authors.forename: C
authors.initials: C
authors.lastname: Purcarea
authors.forename: G
authors.initials: G
authors.lastname: Hervé
authors.forename: R
authors.initials: R
authors.lastname: Cunin
authors.forename: D R
authors.initials: DR
authors.lastname: Evans
mesh_terms: D000595:Amino Acid Sequence; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D019731:Carbon-Nitrogen Ligases