title: Insight into a strategy for attenuating AmpC-mediated beta-lactam resistance: structural basis for selective inhibition of the glycoside hydrolase NagZ.
abstract: NagZ is an exo-N-acetyl-beta-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 beta-lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo-N-acetyl-beta-glucosaminidases: O-GlcNAcase and the beta-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 beta-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 beta-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 beta-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 beta-lactam resistance.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Misty D
authors.initials: MD
authors.lastname: Balcewich
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.forename: Yuan
authors.initials: Y
authors.lastname: He
authors.forename: Terrence W
authors.initials: TW
authors.lastname: James
authors.forename: Gideon J
authors.initials: GJ
authors.lastname: Davies
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D010091:Oximes / Q000737:chemistry* / Q000378:metabolism; D048448:Phenylcarbamates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D010091:Oximes; D048448:Phenylcarbamates; D011993:Recombinant Fusion Proteins; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D001619:beta-N-Acetylhexosaminidases; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D000117:Acetylglucosamine