Looking at Metabolic Regulation and Inborn Errors from a Structural Viewpoint: a Urea Cycle Example

Comunicación presentada en 2nd FEBS Fellows' Meeting, celebrado del 27 al 30 de agosto de 2014 en París (Francia)The urea cycle gets rid of the ammonia derived from protein catabolism, which is highly neurotoxic. Carbamoyl phosphate synthetase 1 (CPS1), a large (1462 residues) six-domain enzyme catalyzing a three-step reaction involving two separate phosphorylation centers, is a key urea cycle catalyst and controller. lt is inactive in the absence of its allosteric activator Nacetyi-L-glutamate (NAG), which is an on/off switch used to prevent amino acid depletion. CPS1 experiences multiple lysine acylation, and deacylation by sirtuin 5, connecting urea cycle control and age-control machinery. >120 CPS1 missense mutations have been reported in patients with CPS1 deficiency (CPS1 D), a urea cycle disorder causing hyperammonemia leading to mental retardation or even to death. Using a baculovirus/insect cell system we have produced pure recombinant human CPS1, which has allowed us to crystallize the enzyme and to determine its structure at 2.4 A-resolution, in ligand-free and NAG and ADP/Pi-bound forms, allowing to understand NAG activation and to place on firm ground our understanding of CPS 1 D, while opening the way for understanding the effects of acylation. Thus, the structure with ligands 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, which account for the lack of usage of glutamine and the utilization of ammonia with high affinity by this enzyme. Furthermore, they help rationalize the disease-causing role of most clinical CPS1 mutations.Supported by Fundación Alicia Koplowitz and Valencian (Prometeo 2009/051) and Spanish (BFU2011-30407; FPU to CO-F) governmentsPeer Reviewe