1DJA

STRUCTURE OF BETA-LACTAMASE PRECURSOR, K73H MUTANT, AT 298K


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Work: 0.168 
  • R-Value Observed: 0.168 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1.

Chen, C.C.Smith, T.J.Kapadia, G.Wasch, S.Zawadzke, L.E.Coulson, A.Herzberg, O.

(1996) Biochemistry 35: 12251-12258

  • DOI: https://doi.org/10.1021/bi961153v
  • Primary Citation of Related Structures:  
    1DJA, 1DJB, 1DJC

  • PubMed Abstract: 

    Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.


  • Organizational Affiliation

    Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
BETA-LACTAMASE258Staphylococcus aureusMutation(s): 1 
Gene Names: BLAZ
EC: 3.5.2.6
UniProt
Find proteins for P00807 (Staphylococcus aureus)
Explore P00807 
Go to UniProtKB:  P00807
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00807
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.90 Å
  • R-Value Work: 0.168 
  • R-Value Observed: 0.168 
  • Space Group: I 2 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 53.9α = 90
b = 93.6β = 90
c = 139.1γ = 90
Software Package:
Software NamePurpose
X-PLORmodel building
X-PLORrefinement
XENGENdata reduction
XENGENdata scaling
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1997-03-12
    Type: Initial release
  • Version 1.1: 2008-03-21
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2011-11-16
    Changes: Atomic model
  • Version 1.4: 2021-11-03
    Changes: Database references, Other
  • Version 1.5: 2024-02-07
    Changes: Data collection