4NP7

Structure of phosphotriesterase mutant (S308L/Y309A) from Agrobacterium radiobacter with diethyl thiophosphate bound in the active site


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.99 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.206 
  • R-Value Observed: 0.208 

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This is version 1.4 of the entry. See complete history


Literature

A 5000-fold increase in the specificity of a bacterial phosphotriesterase for malathion through combinatorial active site mutagenesis

Naqvi, T.Warden, A.C.French, N.Sugrue, E.Carr, P.D.Jackson, C.J.Scott, C.

(2014) PLoS One 9: e94177-e94177

  • DOI: https://doi.org/10.1371/journal.pone.0094177
  • Primary Citation of Related Structures:  
    3WML, 4NP7

  • PubMed Abstract: 

    Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in kcat/KM for malathion. X-ray crystal structures for the PTEAr Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.


  • Organizational Affiliation

    Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan; Ecosystem Sciences, Commonwealth Scientific and Industrial Research Organisation, Canberra, Australian Capital Territory, Australia.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Phosphotriesterase329Agrobacterium tumefaciensMutation(s): 2 
Gene Names: opdA
EC: 3.1.8.1
UniProt
Find proteins for Q93LD7 (Rhizobium radiobacter)
Explore Q93LD7 
Go to UniProtKB:  Q93LD7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ93LD7
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
KCX
Query on KCX
A
L-PEPTIDE LINKINGC7 H14 N2 O4LYS
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.99 Å
  • R-Value Free: 0.251 
  • R-Value Work: 0.206 
  • R-Value Observed: 0.208 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 109.134α = 90
b = 109.134β = 90
c = 63.428γ = 120
Software Package:
Software NamePurpose
MAR345dtbdata collection
MOLREPphasing
REFMACrefinement
XDSdata reduction
Aimlessdata scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2014-09-24
    Type: Initial release
  • Version 1.1: 2015-02-04
    Changes: Other
  • Version 1.2: 2019-11-20
    Changes: Database references, Derived calculations
  • Version 1.3: 2023-11-08
    Changes: Data collection, Database references, Derived calculations, Refinement description
  • Version 1.4: 2023-12-06
    Changes: Data collection