1XX2

Refinement of P99 beta-lactamase from Enterobacter cloacae


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.88 Å
  • R-Value Free: 0.214 
  • R-Value Work: 0.175 

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This is version 1.6 of the entry. See complete history


Literature

Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a beta-lactamase transition-state analog.

Lobkovsky, E.Billings, E.M.Moews, P.C.Rahil, J.Pratt, R.F.Knox, J.R.

(1994) Biochemistry 33: 6762-6772

  • DOI: https://doi.org/10.1021/bi00188a004
  • Primary Citation of Related Structures:  
    1BLS, 1XX2

  • PubMed Abstract: 

    The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.


  • Organizational Affiliation

    Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Beta-lactamase
A, B
361Enterobacter cloacaeMutation(s): 0 
EC: 3.5.2.6
UniProt
Find proteins for P05364 (Enterobacter cloacae)
Explore P05364 
Go to UniProtKB:  P05364
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP05364
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.88 Å
  • R-Value Free: 0.214 
  • R-Value Work: 0.175 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 46.5α = 90
b = 83.47β = 90.01
c = 95.46γ = 90
Software Package:
Software NamePurpose
FRAMBOdata collection
X-GENdata reduction
PHASESphasing
CNSrefinement
X-GENdata scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-11-23
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2014-03-12
    Changes: Database references
  • Version 1.4: 2017-10-11
    Changes: Refinement description
  • Version 1.5: 2021-10-13
    Changes: Database references
  • Version 1.6: 2023-08-23
    Changes: Data collection, Refinement description