title: Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β-lactam antibiotic resistance.
abstract: NagZ is an N-acetyl-β-d-glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram-negative bacteria by removing N-acetyl-glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6-anhydromuramoyl-peptide products generated by NagZ activate β-lactam resistance in many Gram-negative bacteria by inducing the expression of AmpC β-lactamase. Blocking NagZ activity can thereby suppress β-lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X-ray structures of NagZ bound to the potent yet non-selective N-acetyl-β-glucosaminidase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate), and two NagZ-selective inhibitors - EtBuPUG, a PUGNAc derivative bearing a 2-N-ethylbutyryl group, and MM-156, a 3-N-butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM-156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N-acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada, R3T2N2.
authors.forename: Grishma
authors.initials: G
authors.lastname: Vadlamani
authors.affiliation: School of Molecular Sciences, University of Western Australia, Crawley, Western Australia, 6009, Australia.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.affiliation: IC2MP, UMR CNRS 7285, Équipe "Synthèse Organique" Groupe Glycochimie, Université de Poitiers, 4 rue Michel Brunet, 86073, Poitiers cedex 9, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Désiré
authors.affiliation: IC2MP, UMR CNRS 7285, Équipe "Synthèse Organique" Groupe Glycochimie, Université de Poitiers, 4 rue Michel Brunet, 86073, Poitiers cedex 9, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Blériot
authors.affiliation: Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada, V5S 1P6.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada, R3T2N2.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D010091:Oximes / Q000737:chemistry*; D048448:Phenylcarbamates / Q000737:chemistry*; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D018440:beta-Lactam Resistance
chemical_list: D029968:Escherichia coli Proteins; D010091:Oximes; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D006026:Glycoside Hydrolases; D000117:Acetylglucosamine
keywords: AmpC; EtBuPUG; GH3; N-acetyl-β-d-glucosaminidase; NagZ; PUGNAc; family 3; trihydroxyazepane