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, 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.
Vandemeulebroucke
An
A
Department of Molecular and Cellular Interactions and Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Minici
Claudia
C
Bruno
Ilaria
I
Muzzolini
Laura
L
Tornaghi
Paola
P
Parkin
David W
DW
Versées
Wim
W
Steyaert
Jan
J
Degano
Massimo
M
D000818:Animals; D018360:Crystallography, X-Ray; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D009705:Nucleosides / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011688:Purinones / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D009705:Nucleosides; D015800:Protozoan Proteins; D011688:Purinones; D009699:N-Glycosyl Hydrolases