Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-Å crystal structure

Research output: Contribution to journalArticle

96 Scopus citations

Abstract

Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti- parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl β-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca2+ ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k(cat) for the L. major enzyme with inosine, adenosine, guanosine, uridine, and 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 substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.

Original languageEnglish (US)
Pages (from-to)21114-21120
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number30
DOIs
StatePublished - Jul 23 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-Å crystal structure'. Together they form a unique fingerprint.

  • Cite this