The inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic, crystallization, and functional studies

Abstract

The purine salvage pathway is an important target for anti-protozoal drug design due to the absence of de novo synthetic routes in parasitic protozoans. IU-nucleoside hydrolase (IU-NH) catalyzes the hydrolysis of nucleosides through an oxy-carbenium transition state. The attribution of functionality in an enzyme's active-site can be achieved through the determination its three-dimensional structure by x-ray crystallography. The gene for IU-NH has been cloned from genomic DNA by reverse-transcription/PCR of polyA m-RNA from C. fasciculata, using an approach based on the mini-exon sequence and oligo-nucleotides for internal peptides. The primary structure consist of 315 amino acids and agrees with the predicted {dollar}\rm M\sb{lcub}r{rcub} = 34,194{dollar} Da. There is significant homology (an average of 37% in identity) with open reading frames from a wide variety of species, including yeast and a homolog from the human pathogen, Leishmania major. The recombinant enzyme produced by over-expression in E. coli cells, and native enzyme, have both been successfully purified to homogeneity and crystallized. The three-dimensional structure of the enzyme is reminiscent of a nucleotide binding protein, with a core region similar to a Rossmann fold. Conserved clusters of aspartic acid residues are involved in transition state stabilization and binding of a {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar}-metal ion. Site-directed mutagenesis has identified His241 as the proton donor, Asp10 as a metal-ion ligand and the possible active-site base, and Glu166 as a residue crucial for catalysis. The structural information from C. fasciculata will identify targets in other pathogenic protozoa, and will be essential in improving the affinity of inhibitors for IU-nucleoside hydrolase.