Characterization, localization, essentiality and high-resolution crystal structure of Glucosamine 6-phosphate N-Acetyltransferase from Trypanosoma brucei

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

EUKARYOTIC CELL

AMER SOC MICROBIOLOGY

Año: 2011 vol. 10 p. 985 - 997

1535-9778

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (EC 2.3.1.4, TbGNA1) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active and its high-resolution crystal structure obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under non-permissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under non-permissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG) was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African Sleeping Sickness are discussed.