Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance |
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| Authors: | Grishma Vadlamani Keith A Stubbs Jérôme Désiré Yves Blériot David J Vocadlo Brian L Mark |
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| Affiliation: | 1. Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada;2. School of Molecular Sciences, University of Western Australia, Crawley, Western Australia, Australia;3. IC2MP, UMR CNRS 7285, équipe “Synthèse Organique” Groupe Glycochimie, Université de Poitiers, Poitiers cedex 9, France;4. Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada |
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| 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. |
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| Keywords: | NagZ N‐acetyl‐β ‐d‐glucosaminidase GH3 family 3 AmpC PUGNAc EtBuPUG trihydroxyazepane |
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