1I5Q

CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE MUTANT N152A COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.206 
  • R-Value Work: 0.168 

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This is version 1.5 of the entry. See complete history


Literature

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Trehan, I.Beadle, B.M.Shoichet, B.K.

(2001) Biochemistry 40: 7992-7999

  • DOI: https://doi.org/10.1021/bi010641m
  • Primary Citation of Related Structures:  
    1I5Q

  • PubMed Abstract: 

    Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.


  • Organizational Affiliation

    Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Northwestern University, 303 East Chicago Avenue, Chicago, Illinois 60611-3008, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
BETA-LACTAMASE
A, B
358Escherichia coliMutation(s): 1 
Gene Names: AMPC
EC: 3.5.2.6
UniProt
Find proteins for P00811 (Escherichia coli (strain K12))
Explore P00811 
Go to UniProtKB:  P00811
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00811
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
MOX
Query on MOX

Download Ideal Coordinates CCD File 
C [auth A],
D [auth B]
(2R)-2-[(1R)-1-{[(2S)-2-carboxy-2-(4-hydroxyphenyl)acetyl]amino}-1-methoxy-2-oxoethyl]-5-methylidene-5,6-dihydro-2H-1,3-oxazine-4-carboxylic acid
C18 H18 N2 O9
GOYCBKVVHGALFQ-RZAIGCCYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.83 Å
  • R-Value Free: 0.206 
  • R-Value Work: 0.168 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 117.661α = 90
b = 77.267β = 116.75
c = 97.677γ = 90
Software Package:
Software NamePurpose
CNSrefinement
DENZOdata reduction
SCALEPACKdata scaling
CNSphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2001-06-20
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2011-08-24
    Changes: Non-polymer description
  • Version 1.4: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.5: 2023-08-09
    Changes: Data collection, Refinement description