title: Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target.
abstract: The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.
authors.affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Fang
authors.forename: Ting
authors.initials: T
authors.lastname: Du
authors.forename: Olawale G
authors.initials: OG
authors.lastname: Raimi
authors.forename: Ramon
authors.initials: R
authors.lastname: Hurtado-Guerrero
authors.forename: Michael D
authors.initials: MD
authors.lastname: Urbaniak
authors.forename: Adel F M
authors.initials: AF
authors.lastname: Ibrahim
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
authors.forename: Cheng
authors.initials: C
authors.lastname: Jin
authors.forename: Daan M F
authors.initials: DM
authors.lastname: van Aalten
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D002473:Cell Wall / Q000648:ultrastructure; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D020043:Genes, Essential; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; C026146:N-acetylglucosamine-1-phosphate; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D000117:Acetylglucosamine