+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

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



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



Journal of Biological Chemistry 274(30): 21114-21120



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 beta-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 Ca(2+) 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.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 021450278

Download citation: RISBibTeXText

PMID: 10409664

DOI: 10.1074/jbc.274.30.21114


Related references

Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-ANG crystal structure. Journal of Biological Chemistry 274(30): 21114-21120, 1999

Nucleoside hydrolase from Leishmania major Cloning, expression, catalytic properties, transition state inhibitors, and the 25-Angstrom crystal structure. Journal of Biological Chemistry 274.30 (July 23): 21114-21120, 1999

A new class of C-nucleoside analogues 1- -aryl-1,4-dideoxy-1,4-imino-D-ribitols, transition state analogue inhibitors of nucleoside hydrolase. Tetrahedron Letters 34(45): 7213-7224, 1993

Transition-state inhibitors for nucleoside hydrolase and transferase reactions. Official Gazette of the United States Patent & Trademark Office Patents 1238(3), 2000

Amidrazone Analogs of D-Ribofuranose as Transition-State Inhibitors of Nucleoside Hydrolase. Biochemistry 33(13): 3994-4000, 1994

Amidrazone analogues of D-ribofuranose as transition-state inhibitors of nucleoside hydrolase. Biochemistry 33(13): 3994-4000, 1994

Protection of susceptible BALB/c mice from challenge with Leishmania major by nucleoside hydrolase, a soluble exo-antigen of Leishmania. American Journal of Tropical Medicine and Hygiene 77(6): 1060-1065, 2007

Nucleoside hydrolase inhibitor design based on transition state structure. FASEB Journal 7(7): A1199, 1993

Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor. Biochemistry 37(18): 6277-6285, 1998

Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties. Human Vaccines and Immunotherapeutics 12(7): 1707-1720, 2016

Cloning and characterization of Leishmania donovani inosine uridine nucleoside hydrolase. Memorias do Instituto Oswaldo Cruz 94(Suppl. 2): 151, 1999

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies. Proceedings of the National Academy of Sciences of the United States of America 92(25): 11721-5, 1995

Kinetics and docking studies of two potential new inhibitors of the nucleoside hydrolase from Leishmania donovani. European Journal of Medicinal Chemistry 56: 301-307, 2012

Design of inhibitors for nucleoside hydrolase from Leishmania donovani using molecular dynamics studies. Journal Of The Brazilian Chemical Society: 1, 64-73, 2008

Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene. International Journal of Antimicrobial Agents 29(1): 103-107, 2007