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Human carbamoyl phosphate synthetase: structure, function and pathology

AuthorsRubio, Vicente ; Cima, Sergio de; Polo, Luis Mariano ; Díez-Fernández, Carmen ; Cervera, Javier; Fita, Ignacio
Issue Date19-Jul-2014
CitationICAP2014, 24th International Conference on Arginine and Pyrimidines (2014)
AbstractAbstract: TUE 420; pp: 570. Carbamoyl phosphate synthesis from bicarbonate, ammonia and two molecules of ATP, catalyzed by carbamoyl phosphate synthetase 1 (CPS1), is the first step of of the urea cycle, which detoxifies the ammonia produced in protein catabolism. CPS1, a large (1462-residue) multidomain protein having two ATP-binding phosphorylation sites, is very abundant in liver mitocondria (20% of the matrix protein), and is inactive in the absence of the allosteric activator N-acetyl-L-glutamate (NAG). CPS1 deficiency (CPS1D) is an inborn error causing hyperammonemia leading to death or mental retardation. The report of CPS1 regulation by multiple lysine acylation and by deacylation by sirtuin 5 connected the urea cycle with the age-control machinery (Nakagawa et al. Cell 2009; 137:560). The only structure known for a CPS was that of the Escherichia coli enzyme, which only has 40% sequence identity with CPS1 and differs from it in key traits such as the use of glutamine instead of ammonia as preferred substrate (CPS1 cannot use glutamine), its insensitivity to NAG, and for being active in the absence of effectors. Thus, determination of the CPS1 structure appeared essential for understanding CPS1 function and its control by the NAG switch (an extreme case of allosteric activation) and by acylation, and to judge about the pathogenicity of the >130 CPS1 missense mutations reported in CPS1 deficiency. Exploiting our recent baculovirus/insect cell system for recombinant human CPS1 production (Diez-Fernandez et al., Hum Mutat. 2013; 34:1149¿59), we have crystallized the human enzyme and determined its X-ray structure at up to 2.4 A-resolution, in apo and ligand-bound (NAG and ADP/Pi) forms. The liganded structure revealed how does NAG bind in a pocket of the C-terminal domain and has identified elements that are stabilized by ADP binding, as well as conformational changes induced by NAG and ADP binding that lead to define the carbamate tunnel, which in the apo form is heavily branched and open to the environment. Our structures decipher the CPS1 inability to use glutamine and reveal a potential channel for ammonia intake. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations. Supported by Fundacion Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CD-F) governments
DescriptionComunicación presentada en ICAP2014, 24th International Conference on Arginine and Pyrimidines, organizado del 16 al 18 de julio de 2014 en Oxford (Reino Unido)
Abstract publicado en FEBS Journal 281(Supl.1): 570-571 (2014)
Appears in Collections:(IBMB) Comunicaciones congresos
(IBV) Comunicaciones congresos
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