3FKW

AmpC K67R mutant apo structure


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.166 
  • R-Value Observed: 0.167 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Re-examining the role of Lys67 in class C beta-lactamase catalysis.

Chen, Y.McReynolds, A.Shoichet, B.K.

(2009) Protein Sci 18: 662-669

  • DOI: https://doi.org/10.1002/pro.60
  • Primary Citation of Related Structures:  
    3FKV, 3FKW

  • PubMed Abstract: 

    Lys67 is essential for the hydrolysis reaction mediated by class C beta-lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C beta-lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2- to 3-fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C beta-lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61-fold decrease in k(cat), the deacylation rate. We have determined the structure of the K67R mutant by X-ray crystallography both in apo and transition state-analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C beta-lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.


  • Organizational Affiliation

    Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158-2550, 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: ampAampCb4150JW4111
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
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.199 
  • R-Value Work: 0.166 
  • R-Value Observed: 0.167 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 118.348α = 90
b = 76.564β = 115.82
c = 97.702γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
REFMACrefinement
PDB_EXTRACTdata extraction
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
REFMACphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-03-24
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2017-11-01
    Changes: Refinement description
  • Version 1.3: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-09-06
    Changes: Data collection, Refinement description