3P62

Wild-type pentaerythritol tetranitrate reductase containing a C-terminal 8-histidine tag


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.156 
  • R-Value Work: 0.126 
  • R-Value Observed: 0.127 

Starting Model: experimental
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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site-Saturated Libraries

Hulley, M.E.Toogood, H.S.Fryszkowska, A.Mansell, D.Stephens, G.M.Gardiner, J.M.Scrutton, N.S.

(2010) Chembiochem 11: 2433-2447

  • DOI: https://doi.org/10.1002/cbic.201000527
  • Primary Citation of Related Structures:  
    3P62, 3P67

  • PubMed Abstract: 

    This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.


  • Organizational Affiliation

    Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Pentaerythritol tetranitrate reductase373Enterobacter cloacaeMutation(s): 0 
Gene Names: onrPETNR
EC: 1.6.99.1
UniProt
Find proteins for P71278 (Enterobacter cloacae)
Explore P71278 
Go to UniProtKB:  P71278
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP71278
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Free: 0.156 
  • R-Value Work: 0.126 
  • R-Value Observed: 0.127 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 58.961α = 90
b = 68.954β = 90
c = 88.476γ = 90
Software Package:
Software NamePurpose
SCALAdata scaling
MOLREPphasing
REFMACrefinement
PDB_EXTRACTdata extraction
ADSCdata collection
MOSFLMdata reduction

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2010-11-24
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2023-11-01
    Changes: Data collection, Database references, Derived calculations, Refinement description