The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus , a missing link in the evolution of carbamoyl phosphate biosynthesis
- 25 November 1997
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 94 (24), 12803-12808
- https://doi.org/10.1073/pnas.94.24.12803
Abstract
Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100°C or 3 h at 95°C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.Keywords
This publication has 33 references indexed in Scilit:
- Structure of Carbamoyl Phosphate Synthetase: A Journey of 96 Å from Substrate to Product,Biochemistry, 1997
- Function of the Major Synthetase Subdomains of Carbamyl-phosphate SynthetaseJournal of Biological Chemistry, 1996
- Archaeal transcription factors and their role in transcription initiationFEMS Microbiology Reviews, 1996
- Purification and Characterization of Carbamoyl‐Phosphate Synthetase from the Deep‐Sea Hyperthermophilic Archaebacterium Pyrococcus abyssiEuropean Journal of Biochemistry, 1996
- Ammonia-dependent synthesis and metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosusMicrobiology, 1995
- Carbamyl Phosphate Synthetase III, an Evolutionary Intermediate in the Transition Between Glutamine-dependent and Ammonia-dependent Carbamyl Phosphate SynthetasesJournal of Molecular Biology, 1994
- Crystallization, Characterization and Preliminary Crystallographic Studies of Carbamate Kinase of Streptococcus faeciumJournal of Molecular Biology, 1994
- Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunitBiochemistry, 1992
- A novel genetic system to detect protein–protein interactionsNature, 1989
- Mechanism and Regulation of the Glutamine‐Dependent Carbamyl Phosphate Synthetase of Escherichia ColiPublished by Wiley ,1989