Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N -Acetyltransferase from Trypanosoma brucei

Abstract

A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was 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 nonpermissive 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 nonpermissive 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.

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

“…Short-term interruption already leads to arrest of growth in heterotrophic organisms when no external GlcNAc source is supplied as was shown for Trypanosoma brucei [9]. Short-term interruption already leads to arrest of growth in heterotrophic organisms when no external GlcNAc source is supplied as was shown for Trypanosoma brucei [9].…”

Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages

“…cruzi ( S1 Fig ) and have recently been shown to be essential for blood stage T . brucei [ 17 ]. The targeted deletion of the L .…”

Identification of a glycosylphosphatidylinositol anchor-modifying β1-3 galactosyltransferase in Trypanosoma brucei

“…The survival strategies of protozoan parasites frequently involve the participation of glycoconjugates. In the particular case of T. brucei , several precursors required for the biosynthesis of these glycoconjugates such as UDP-Gal and UDP-GlcNAc ( Roper et al 2002 , 2005 ; Stokes et al 2008 ; Marino et al 2011 ) are essential for the survival of the parasite in the bloodstream and/or procyclic form life stages. This prompted us to mine the T. brucei genome for UDP-sugar-dependent GTs and to investigate their essentiality, function and specificity by the creation of null or conditional null mutants.…”

“…The creation of UDP-glucose 4′-epimerase ( TbGalE ) conditional null mutants showed that this gene, and hence UDP-Gal, is essential for the survival of the parasite in both the bloodstream and procyclic life stages ( Roper et al 2002 ; Roper et al 2005 ; Urbaniak et al 2006 ). Similarly, the creation of UDP-GlcNAc pyrophosphorylase ( TbUAP ) and glucosamine 6-phosphate N -acetyltransferase ( TbGNA1 ) conditional null mutants has shown that UDP-GlcNAc is also essential for bloodstream form T. brucei ( Marino et al 2011 ; Stokes et al 2008 ). From these experiments, it is possible to conclude that one or more of the UDP-Gal- and UDP-GlcNAc-dependent glycosylation pathways are essential to the parasite.…”