Expresión y estudio de estructura-función de N-acetilglutamato sintasa humana, diana de un error congénito del ciclo de la urea

Abstract

[ES] Este trabajo está enfocado al estudio de N-acetilglutamato sintasa (NAGS) humana, enzima del ciclo de la urea cuyo déficit causa acumulación de amonio, que es un potente neurotóxico que al acumularse puede dar lugar al coma e incluso a la muerte. NAGS es un enzima muy inestable que no puede producirse con facilidad de manera recombinante. Hasta el momento, el laboratorio donde realizaré este trabajo produce NAGS humana asociada a la proteína de unión a maltosa. Con esta construcción han caracterizado cinéticamente este enzima, habiendo estudiado el impacto de algunas mutaciones clínicas. Sin embargo, la presencia de una etiqueta tan grande (42 kDa) limita la posibilidad de llevar a cabo estudios estructurales. Nos planteamos con este trabajo producir una forma más sencilla de NAGS humana estable. Para ello sustituiremos la proteína de unión a maltosa por una etiqueta de polihistidinas y emplearemos estrategias para favorecer la solubilidad de la proteína. Resultados previos obtenidos por un grupo colaborador apoyan que tendremos éxito en la sobreexpresión de esta forma de HuNAGS, que purificaremos a gran escala utilizando cromatografía de afinidad y exclusión molecular. Tras la purificación del enzima, lo caracterizaremos desde el punto de vista de su estabilidad y su actividad enzimática, estableciendo sus parámetros cinéticos y comparándolos con los previamente obtenidos en el laboratorio para la proteína fusionada a MBP, estudiando su forma oligomérica, y poniendo a punto sus condiciones de concentración con el fin de poder iniciar estudios estructurales mediante cristalografía de rayos X y microscopia electrónica de partícula individual.

[EN] N-acetyl-L-glutamate synthase (NAGS) is the enzyme that catalyzes the acetylation of Lglutamate using acetyl coenzyme A to produce N-acetyl-L-glutamate (NAG). In mammals NAG plays a crucial role in the proper functioning of the urea cycle, a pathway that serves to remove ammonium resulting from protein catabolism, converting it into urea, which is a harmless molecule that can be eliminated through urine. NAG is the essential activator of the first enzyme of the urea cycle, carbamoyl phosphate synthetase I (CPS1), in the absence of which this enzyme is practically inactive. Congenital N-acetyl-L-glutamate synthase deficiency (NAGSD; OMIM #237310) is the rarest disorder of the urea cycle, being clinically indistinguishable from CPS1 deficiency. Both result in ammonium accumulation, leading to neurological damage although only NAGSD is treatable, by administering carbamylglutamate, which is an analog of NAG that also activates CPS1 allowing urea cycle function and thus ammonium elimination. Human NAGS is a very unstable enzyme of which little is known at the molecular level. Its functional and structural characterization would allow addressing the challenges it presents, which are mainly the discrimination between pathogenic and trivial variants, the understanding of its activation by arginine, the establishment of the basis of the extraordinary instability and the discovery of stabilization forms that would allow the identification and study of destabilizing mutations. Previous studies in Dr. Vicente Rubio's laboratory were able to stabilize human NAGS (HuNAGS) by introducing the maltose-binding protein (MBP) at its amino-terminal end, which allowed the fusion protein to be expressed in Escherichia coli and purified in abundance. However, the presence of such a large tag (MBP) limited the possibility of structural studies and when they tried to separate MBP from HuNAGS, the latter precipitated. Therefore, in this work we set out to obtain a simpler form of soluble, pure, homogeneous, active and stable HuNAGS, which would allow us to initiate studies to determine the structure of this enzyme. Thus, we have overexpressed in Escherichia coli the conserved region of HuNAGS fused to a label with six histidines in its amino terminal region, which facilitates its purification, and we have optimized the conditions to obtain soluble, pure and homogeneous recombinant HuNAGS. We have studied the stability of the purified HuNAGS, its oligomeric state and its enzymatic activity and kinetic parameters, having initiated structural studies by X-ray crystallography and single particle electron cryomicroscopy.