👤 Satu Pekkala

The urea cycle disease carbamoyl-phosphate synthetase deficiency (CPS1D) has been associated with many mutations in the CPS1 gene [Häberle et al., 2011. Hum Mutat 32:579-589]. The disease-causing potential of most of these mutations is unclear. To test the mutations effects, we have developed a system for recombinant expression, mutagenesis, and purification of human carbamoyl-phosphate synthetase 1 (CPS1), a very large, complex, and fastidious enzyme. The kinetic and molecular properties of recombinant CPS1 are essentially the same as for natural human CPS1. Glycerol partially replaces the essential activator N-acetyl-l-glutamate (NAG), opening possibilities for treating CPS1D due to NAG site defects. The value of our expression system for elucidating the effects of mutations is demonstrated with eight clinical CPS1 mutations. Five of these mutations decreased enzyme stability, two mutations drastically hampered catalysis, and one vastly impaired NAG activation. In contrast, the polymorphisms p.Thr344Ala and p.Gly1376Ser had no detectable effects. Site-limited proteolysis proved the correctness of the working model for the human CPS1 domain architecture generally used for rationalizing the mutations effects. NAG and its analogue and orphan drug N-carbamoyl-l-glutamate, protected human CPS1 against proteolytic and thermal inactivation in the presence of MgATP, raising hopes of treating CPS1D by chemical chaperoning with N-carbamoyl-l-glutamate. Show less

Carbamoyl phosphate synthetase 1 (CPS1) plays a paramount role in liver ureagenesis since it catalyzes the first and rate-limiting step of the urea cycle, the major pathway for nitrogen disposal in humans. CPS1 deficiency (CPS1D) is an autosomal recessive inborn error which leads to hyperammonemia due to mutations in the CPS1 gene, or is caused secondarily by lack of its allosteric activator NAG. Proteolytic, immunological and structural data indicate that human CPS1 resembles Escherichia coli CPS in structure, and a 3D model of CPS1 has been presented for elucidating the pathogenic role of missense mutations. Recent availability of CPS1 expression systems also can provide valuable tools for structure-function analysis and pathogenicity-testing of mutations in CPS1. In this paper, we provide a comprehensive compilation of clinical CPS1 mutations, and discuss how structural knowledge of CPS enzymes in combination with in vitro analyses can be a useful tool for diagnosis of CPS1D. Show less

Carbamoyl-phosphate synthetase I (CPS1) deficiency (CPS1D), a recessively inherited urea cycle error due to CPS1 gene mutations, causes life-threatening hyperammonemia. The disease-causing potential of missense mutations in CPS1 deficiency can be ascertained with the recombinant CPS1 expression and purification system reported here, which uses baculovirus and insect cells. We study with this system the effects of nine clinical mutations and one polymorphism on CPS1 solubility, stability, activity, and kinetic parameters for NAG. Five of the mutations (p.T471N, p.Q678P, p.P774L, p.R1453Q, and p.R1453W) are first reported here, in three severe CPS1D patients. p.P774L, p.R1453Q, and p.R1453W inactivate CPS1, p.T471N and p.Y1491H greatly decrease the apparent affinity for NAG, p.Q678P hampers correct enzyme folding, and p.S123F, p.H337R, and p.P1411L modestly decrease activity. p.G1376S is confirmed a trivial polymorphism. The effects of the C-terminal domain mutations are rationalized in the light of this domain crystal structure, including the NAG site structure [Pekkala et al. Biochem J 424:211-220]. The agreement of clinical observations and in vitro findings, and the possibility to identify CPS1D patients who might benefit from specific treatment with NAG analogues because they exhibit reduced affinity for NAG highlight the value of this novel CPS1 expression/purification system. Show less