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author:("Fang, wenxin")
1.  Genetic and structural validation of Aspergillus fumigatus N-acetylphosphoglucosamine mutase as an antifungal target 
Bioscience Reports  2013;33(5):e00063.
Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)–GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low μM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.
PMCID: PMC3763426  PMID: 23844980
cell wall; drug target; enzyme; inhibitor; nucleotide sugar; protein structure; AfAGM1, A. fumigatus N-acetylphosphoglucosamine mutase; AGM1, N-acetylphosphoglucosamine mutase; CaAGM1, Candida albicans AGM1; Fru-6P, fructose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GlcNAc, N-acetylglucosamine; GlcNAc-1P, N-acetylglucosamine-1-phosphate; GlcN-6P, glucosamine 6-phosphate; GFA1, glutamine: Fru-6P amidotransferase; GNA1, GlcN-6P acetyltransferase; IA, invasive aspergillosis; MIC, minimum inhibitory concentration; MM, minimal medium; RMSD, root mean square deviation; UAP1, UDP–GlcNAc pyrophosphorylase; UDP, uridine diphosphate
2.  Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1 
Glucosamine-6-phosphate N-acetyltransferase is an essential enzyme of the eukaryotic UDP-GlcNAc biosynthetic pathway. A crystal structure at 1.55 Å resolution revealed a highly unusual covalent product complex and biochemical studies investigated the function of a fully conserved active-site cysteine.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.
PMCID: PMC3413214  PMID: 22868768
carbohydrates; glycobiology; Caenorhabditis elegans; glucosamine-6-phosphate N-acetyltransferase; coenzyme A adduct; mechanism
3.  A Novel Allosteric Inhibitor of the Uridine Diphosphate N-Acetylglucosamine Pyrophosphorylase from Trypanosoma brucei 
ACS Chemical Biology  2013;8(9):1981-1987.
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi–bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP–inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
PMCID: PMC3780468  PMID: 23834437
4.  Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target 
Molecular Microbiology  2013;89(3):479-493.
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.
PMCID: PMC3888555  PMID: 23750903

Results 1-4 (4)