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Título

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

Autor Ramón-Maiques, Santiago ; Marina, Alberto ; Uriarte, Matxalen; Fita, Ignacio ; Rubio, Vicente
Palabras clave ADP site
Arginine metabolism
Hyperthermophiles
Pyrococcus furiosus
Phosphoryl group transfer
Fecha de publicación 2-jun-2000
EditorAcademic Press
Citación Journal of Molecular Biology 299(2): 463-476 (2000)
ResumenCarbamoyl phosphate (CP), an essential precursor of arginine and the pyrirnidine bases, is synthesized by CP synthetase (CPS) in three steps. The last step, the phosphorylation of carbamate, is also catalyzed by carbamate kinase (CK), an enzyme used by microorganisms to produce ATP from ADP and CP. Although the recently determined structures of CPS and CK show no obvious mutual similarities, a CK-like CPS reported in hyperthermophilic archaea was postulated to be a missing link in the evolution of CP biosynthesis. The 1.5 Å resolution structure of this enzyme from Pyrococcus furiosus shows both a subunit topology and a homodimeric molecular organization, with a 16-stranded open β-sheet core surrounded by α-helices, similar to those in CK. However, the pyrococcal enzyme exhibits many solvent-accessible ion-pairs, an extensive, strongly hydrophobic, intersubunit surface, and presents a bound ADP molecule, which does not dissociate at 22°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 ring enclosed in a pocket specific for adenine. Overall, the enzyme structure is ill-suited for catalyzing the characteristic three-step reaction of CPS and supports the view that the CK-like CPS is in fact a highly thermostable and very slow (at 37°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 hydrophobic intersubunit contacts and from the large number of exposed ion-pairs, some of which form ion-pair networks across several secondary structure elements in each enzyme subunit. The structure provides the first information on substrate binding and catalysis in CKs, and suggests that the slow rate at 37°C is possibly a consequence of slow product dissociation. (C) 2000 Academic Press.
Versión del editorhttp://dx.doi.org/10.1006/jmbi.2000.3779
URI http://hdl.handle.net/10261/111189
DOI10.1006/jmbi.2000.3779
Identificadoresdoi: 10.1006/jmbi.2000.3779
issn: 0022-2836
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