Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

Abstract

NagZ is an exo-N-acetyl-b-glucosaminidase, found within Gram-negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N-acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC b-lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo-N-acetyl-b-glucosaminidases: O-GlcNAcase and the b-hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring-group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2-N-acyl derivatives of O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram-negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N-valeryl-PUGNAc, the most selective known inhibitor of NagZ over both the human b-hexosaminidases and O-GlcNAcase. The selectivity stems from the five-carbon acyl chain of N-valeryl-PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O-GlcNAcase homologue bound to a related inhibitor N-butyryl-PUGNAc, which bears a four-carbon chain and is selective for both NagZ and O-GlcNAcase over the human b-hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O-GlcNAcase homologue. A comparison of these complexes, and with the human b-hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2-N-acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated b-lactam resistance.

N-Acetylglucosaminidases from CAZy Family GH3 Are Really Glycoside Phosphorylases, Thereby Explaining Their Use of Histidine as an Acid/Base Catalyst in Place of Glutamic Acid

“…1 Recipient of a Schrodinger Fellowship from the Austrian Science Fund. 2 Supported by a Tier 1 Canada Research Chair. To whom correspondence should be addressed.…”

“…A subset of these enzymes plays a role in the recycling of peptidoglycan by removing the nonreducing end N-acetylglucosamine from the disaccharide product released by lytic transglycosylases and muramidases (GlcNAc-anhydro-MurNAc 3 -peptide and its hydrated version) in Gram-negative and Gram-positive bacteria, respectively (1). The resultant (anhydro)-MurNAc peptide is an activator of ␤-lactamase production in some Gram-negative bacteria, rendering the GH3 N-acetylglucosaminidases a possible therapeutic target (2)(3)(4). Accordingly, considerable attention has been devoted to the generation of potent, selective inhibitors for this group of enzymes.…”

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

“…β-Hexosaminidases from Vibrio cholerae (NagZ; PDB IDs 1TR9 and 1Y65; New York SGX Research Center for Structural Genomics), which has one domain, and β-hexosaminidases from Bacillus subtilis (PDB IDs 3BMX and 3CQM; Konstanz University), which has two domains, have also been deposited in the PDB. The structure of the V. cholerae enzyme has also been determined in complex with three inhibitors 18,19 (PDB IDs 2OXN, 3GS6, and 3GSM). However, only the H. vulgare enzyme has been characterized in detail with regard to its structure, enzymatic mechanism, and substrate specificity.…”

Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus

“…The enzymes catalyse the hydrolysis of O-glucosidic bonds to release Glc (glucose) units from the non-reducing end. β-D-glucan glucohydrolase from Hordeum vulgare (ExoI) [4], two β-N-acetylhexosaminidases from Vibrio cholerae (NagZ) and Bacillus subtilis (YbbD) (PDB code 3BMX) [5,6], exo-1,3-1,4-β-glucanase (ExoP) from Pseudoalteromonas sp. The enzyme was classified in the GH3 (glycoside hydrolase family 3) [2], one of the largest families in the CAZy database (http://www.cazy.org) [3].…”