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

TY - JOUR

T1 - Insight into a strategy for attenuating AmpC-mediated beta-lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

AU - Balcewich, Misty D.

AU - Stubbs, Keith A.

AU - He, Yuan

AU - James, Terrence W.

AU - Davies, Gideon J.

AU - Vocadlo, David J.

AU - Mark, Brian L.

PY - 2009/7

Y1 - 2009/7

N2 - 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.

AB - 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.

KW - NagZ

KW - beta-glucosaminidase

KW - beta-hexosaminidase

KW - O-GlcNAcase

KW - beta-lactam

KW - X-ray crystal structure

KW - glycoside hydrolase

KW - glycosidase

KW - peptidoglycan recycling

KW - SUBSTRATE-ASSISTED CATALYSIS

KW - TAY-SACHS-DISEASE

KW - O-GLCNACASE

KW - N-ACETYLGLUCOSAMINIDASE

KW - ESCHERICHIA-COLI

KW - KLEBSIELLA-PNEUMONIAE

KW - D-GLUCOSAMINIDASE

KW - MECHANISM

KW - PLASMID

KW - HEXOSAMINIDASE

UR - http://www.scopus.com/inward/record.url?scp=67650070343&partnerID=8YFLogxK

U2 - 10.1002/pro.137

DO - 10.1002/pro.137

M3 - Article

SN - 0961-8368

VL - 18

SP - 1541

EP - 1551

JO - Protein Science

JF - Protein Science

IS - 7

ER -