2WYX

Neutron structure of a class A Beta-lactamase Toho-1 E166A R274N R276N triple mutant


Experimental Data Snapshot

  • Method: NEUTRON DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.223 
  • R-Value Observed: 0.226 

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This is version 1.3 of the entry. See complete history


Literature

Neutron Diffraction Studies of a Class a Beta-Lactamase Toho-1 E166A R274N R276N Triple Mutant

Tomanicek, S.J.Blakeley, M.P.Cooper, J.Chen, Y.Afonine, P.Coates, L.

(2010) J Mol Biol 396: 1070

  • DOI: https://doi.org/10.1016/j.jmb.2009.12.036
  • Primary Citation of Related Structures:  
    2WYX

  • PubMed Abstract: 

    beta-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the beta-lactam antibiotics is the production of beta-lactamase enzymes that inactivate beta-lactams by rapidly hydrolyzing the amide group of the beta-lactam ring. Specifically, the class A extended-spectrum beta-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a beta-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.


  • Organizational Affiliation

    Neutron Scattering Science Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
BETA-LACTAMSE TOHO-1256Escherichia coli BL21Mutation(s): 3 
EC: 3.5.2.6
UniProt
Find proteins for Q47066 (Escherichia coli)
Explore Q47066 
Go to UniProtKB:  Q47066
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ47066
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: NEUTRON DIFFRACTION
  • Resolution: 2.10 Å
  • R-Value Free: 0.258 
  • R-Value Work: 0.223 
  • R-Value Observed: 0.226 
  • Space Group: P 32 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 72.993α = 90
b = 72.993β = 90
c = 98.365γ = 120
Software Package:
Software NamePurpose
PHENIXrefinement
LAUEGENdata reduction
LAUEGENdata scaling

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-01-12
    Type: Initial release
  • Version 1.1: 2018-01-31
    Changes: Data collection
  • Version 1.2: 2018-05-09
    Changes: Data collection
  • Version 1.3: 2024-05-08
    Changes: Data collection, Database references, Other