title: 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.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Karina
authors.initials: K
authors.lastname: Mariño
authors.forename: M Lucia Sampaio
authors.initials: ML
authors.lastname: Güther
authors.forename: Amy K
authors.initials: AK
authors.lastname: Wernimont
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Raymond
authors.initials: R
authors.lastname: Hui
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D000595:Amino Acid Sequence; D003461:Crystallography; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D051081:Glucosamine 6-Phosphate N-Acetyltransferase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008830:Microbodies / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011134:Polysaccharides / Q000032:analysis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014353:Trypanosomiasis, African; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000737:chemistry / Q000235:genetics
chemical_list: D011134:Polysaccharides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; C037199:poly-N-acetyllactosamine; D051081:Glucosamine 6-Phosphate N-Acetyltransferase