Nucleoside hydrolase from leishmania major - Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-angstrom crystal structure

Protozoan parasites lack the pathway of the de novo synthesis of purines an d depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the pur ine salvage pathway and represents a prime target for the development of an ti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leis hmania major was cloned and sequence analysis revealed that the L. major nu cleoside hydrolase shares 78% sequence identity with the nonspecific nucleo side hydrolase from Crithidia fasciculata. The L. major enzyme was overexpr essed in Escherichia coli and purified to over 95% homogeneity, The L. majo r nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-n itrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both ino sine and uridine nucleosides as favored substrates; and 3) significant acti vity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a boun d Ca2+ ion in the active site with five oxygen ligands hom Asp-10, Asp-15 ( bidentate), Thr-126 (carbonyl), and Asp-241, The structure is similar to th e C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similaritie s, the catalytic specificities differ substantially. Relative values of k(c at) for the L. major enzyme with inosine, adenosine, guanosine, uridine, an d cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same subst rates. Iminoribitol analogues of the transition state are nanomolar inhibit ors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.