1RCJ

Crystal structure of E166A mutant of SHV-1 beta-lactamase with the trans-enamine intermediate of tazobactam


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.171 
  • R-Value Work: 0.147 

wwPDB Validation   3D Report Full Report


Ligand Structure Quality Assessment 


This is version 1.5 of the entry. See complete history


Literature

Tazobactam Forms a Stoichiometric trans-Enamine Intermediate in the E166A Variant of SHV-1 beta-Lactamase: 1.63 A Crystal Structure

Padayatti, P.S.Helfand, M.S.Totir, M.A.Carey, M.P.Hujer, A.M.Carey, P.R.Bonomo, R.A.Van Den Akker, F.

(2004) Biochemistry 43: 843-848

  • DOI: https://doi.org/10.1021/bi035985m
  • Primary Citation of Related Structures:  
    1RCJ

  • PubMed Abstract: 

    Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M. S., et al. (2003) Biochemistry 42, 13386-13392]. The Raman data show that maximal populations of an enamine-like intermediate occur 20-30 min after "soaking in" has commenced. By flash-freezing crystals in this time frame, we were able to trap the enamine species. The resulting 1.63 A resolution crystal structure revealed tazobactam covalently bound in the trans-enamine intermediate state with close to 100% occupancy in the active site. The Raman data also indicated that tazobactam forms a larger population of enamine than sulbactam or clavulanic acid does and that tazobactam's intermediate is also the most long-lived. The crystal structure provides a rationale for this finding since only tazobactam is able to form favorable intra- and intermolecular interactions in the active site that stabilize this trans-enamine intermediate. These interactions involve both the sulfone and triazolyl groups that distinguish tazobactam from clavulanic acid and sulbactam, respectively. The observed stabilization of the transient intermediate of tazobactam is thought to contribute to tazobactam's superior in vitro and in vivo clinical efficacy. Understanding the structural details of differing inhibitor effectiveness can aid the design of improved mechanism-based beta-lactamase inhibitors.


  • Organizational Affiliation

    Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Beta-lactamase SHV-1265Klebsiella pneumoniaeMutation(s): 1 
Gene Names: BLASHV1
EC: 3.5.2.6
UniProt
Find proteins for P0AD64 (Klebsiella pneumoniae)
Explore P0AD64 
Go to UniProtKB:  P0AD64
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0AD64
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.171 
  • R-Value Work: 0.147 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 49.848α = 90
b = 55.193β = 90
c = 83.733γ = 90
Software Package:
Software NamePurpose
SCALEPACKdata scaling
CNSrefinement
CNSphasing

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-02-17
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Refinement description
  • Version 1.4: 2021-10-27
    Changes: Database references, Derived calculations
  • Version 1.5: 2023-08-23
    Changes: Data collection, Refinement description