Structure and Mechanism of the 6-Oxopurine Nucleosidase from Trypanosoma brucei brucei

Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes,...

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Published in:Biochemistry (Easton) 2010-10, Vol.49 (41), p.8999-9010
Main Authors: Vandemeulebroucke, An, Minici, Claudia, Bruno, Ilaria, Muzzolini, Laura, Tornaghi, Paola, Parkin, David W, Versées, Wim, Steyaert, Jan, Degano, Massimo
Format: Article
Language:English
Subjects:
Animals
Crystallography, X-Ray
Kinetics
N-Glycosyl Hydrolases - chemistry
N-Glycosyl Hydrolases - metabolism
Nucleosides - chemistry
Nucleosides - metabolism
Protozoan Proteins - chemistry
Protozoan Proteins - metabolism
Purinones - chemistry
Purinones - metabolism
Structure-Activity Relationship
Trypanosoma brucei brucei - enzymology
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Summary:Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes, although they are thought to play a primary role in the catabolism of exogenously derived nucleosides. Here, we report the first functional and structural characterization of the inosine-guanosine-specific NH from Trypanosoma brucei brucei. The enzyme shows near diffusion-limited efficiency coupled with a clear specificity for 6-oxopurine nucleosides achieved through a catalytic selection of these substrates. Pre-steady-state kinetic analysis reveals ordered product release, and a rate-limiting structural rearrangement that is associated with the release of the product, ribose. The crystal structure of this trypanosomal NH determined to 2.5 Å resolution reveals distinctive features compared to those of both purine- and pyrimidine-specific isozymes in the framework of the conserved and versatile NH fold. Nanomolar iminoribitol-based inhibitors identified in this study represent important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi100697d