Identification, subcellular localization, biochemical properties and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase.

MARIÑO, KARINA; SAMPAIO GUTHER, MARIA LUCIA; WERNIMONT, AMY K.; AMANI, MEHRNAZ; HUI, RAYMOND; FERGUSON, M.A.J

GLYCOBIOLOGY

OXFORD UNIV PRESS INC

Año: 2010 vol. 20 p. 1619 - 1630

0959-6658

The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite’s survival andinfectivity and the de novo biosyntheses of the sugar nucleotides UDP-galactose, UDP-Nacetylglucosamine and GDP-fucose have been shown to be essential for the growth. The only route to UDP-galactose in T.brucei is through the epimerization of UDP-glucose by UDP-glucose 4´- epimerase. UDP-glucose is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (b-D-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T.brucei. Considering that UDP-glucose plays such a central role in carbohydrate metabolism, we decided to characterize UDP-glucose biosynthesis in T.brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-glucose from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme.