title: Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: effects of CPS1 mutations that concentrate in a central domain of unknown function.
abstract: Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased V(max). Substantial although not dramatic increases in K(m) values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations. Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of "Integrating Domain" for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions.
authors.affiliation: Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Díez-Fernández
authors.affiliation: University Children's Hospital, Zurich and Children's Research Center, Zurich, Switzerland.
authors.forename: Liyan
authors.initials: L
authors.lastname: Hu
authors.affiliation: Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain; Group 739 of the Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER) del Instituto de Salud Carlos III, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Cervera
authors.affiliation: University Children's Hospital, Zurich and Children's Research Center, Zurich, Switzerland. Electronic address: johannes.haeberle@kispi.uzh.ch.
authors.forename: Johannes
authors.initials: J
authors.lastname: Häberle
authors.affiliation: Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain; Group 739 of the Centro de Investigación Biomédica en Red sobre Enfermedades Raras (CIBERER) del Instituto de Salud Carlos III, Spain. Electronic address: rubio@ibv.csic.es.
authors.forename: Vicente
authors.initials: V
authors.lastname: Rubio
mesh_terms: D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020165:Carbamoyl-Phosphate Synthase I Deficiency Disease / Q000235:genetics* / Q000473:pathology*; D002460:Cell Line; D004795:Enzyme Stability; D005971:Glutamates / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D005971:Glutamates; D011994:Recombinant Proteins; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid
keywords: CPS 1 structure; CPS1 deficiency; Hyperammonemia; Inborn errors; Site-directed mutagenesis; Urea cycle diseases