Molecular basis of pyrimidine 5'-nucleotidase deficiency caused by 3 newly identified missense mutations (c.187T>C, c.469G>C and c.740T>C) and a tabulation of known mutations.

Abstract Hereditary pyrimidine 5′-nucleotidase deficiency is the most frequent enzymopathy of red blood cell nucleotide metabolism that causes hereditary non-spherocytic hemolytic anemia. The disease is usually characterized by mild-to-moderate hemolytic anemia, reticulocytosis and hyperbilirubinemia. To date, diagnosis ultimately depends upon demonstration of a reduced level of pyrimidine 5′-nucleotidase type-I (P5′N-1) activity in red cells and detection of mutations in the P5′N-1 gene. To unravel the causes of the P5′N deficiency and to obtain data for a definitive diagnosis three newly described missense mutations (c.187T>C, c.469G>C and c.740T>C) identified in patients with hemolytic anemia have been characterized at protein level. The mutant enzymes (C63R, G157R and I247T) were obtained as recombinant forms and purified to homogeneity. The enzymes were altered, although to a different extent, in both thermal stability and catalytic efficiency. The catalytic efficiency of all mutants was reduced especially towards UMP (up to more than 200 times), owing to the increased K m values (approximately, 10–25 times higher). The G157R enzyme was severely heat unstable and lost half of its activity after about 23 min of incubation at 37 °C. At higher temperature C63R and I247T mutants as well were less stable than the wild-type enzyme. Therefore, although the mutations targeted different regions of the P5′N-1 structure, they produced similar effects on the molecular properties of the enzyme. Thus, all affected amino acids are functionally and structurally important for preserving the enzyme activity during the red cell life span.