4IXT

Structure of a 37-fold mutant of halohydrin dehalogenase (HheC) bound to ethyl (R)-4-cyano-3-hydroxybutyrate


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.49 Å
  • R-Value Free: 0.249 
  • R-Value Work: 0.199 
  • R-Value Observed: 0.202 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Biocatalytic and structural properties of a highly engineered halohydrin dehalogenase.

Schallmey, M.Floor, R.J.Hauer, B.Breuer, M.Jekel, P.A.Wijma, H.J.Dijkstra, B.W.Janssen, D.B.

(2013) Chembiochem 14: 870-881

  • DOI: https://doi.org/10.1002/cbic.201300005
  • Primary Citation of Related Structures:  
    4IXT, 4IXW, 4IY1

  • PubMed Abstract: 

    Two highly engineered halohydrin dehalogenase variants were characterized in terms of their performance in dehalogenation and epoxide cyanolysis reactions. Both enzyme variants outperformed the wild-type enzyme in the cyanolysis of ethyl (S)-3,4-epoxybutyrate, a conversion yielding ethyl (R)-4-cyano-3-hydroxybutyrate, an important chiral building block for statin synthesis. One of the enzyme variants, HheC2360, displayed catalytic rates for this cyanolysis reaction enhanced up to tenfold. Furthermore, the enantioselectivity of this variant was the opposite of that of the wild-type enzyme, both for dehalogenation and for cyanolysis reactions. The 37-fold mutant HheC2360 showed an increase in thermal stability of 8 °C relative to the wild-type enzyme. Crystal structures of this enzyme were elucidated with chloride and ethyl (S)-3,4-epoxybutyrate or with ethyl (R)-4-cyano-3-hydroxybutyrate bound in the active site. The observed increase in temperature stability was explained in terms of a substantial increase in buried surface area relative to the wild-type HheC, together with enhanced interfacial interactions between the subunits that form the tetramer. The structures also revealed that the substrate binding pocket was modified both by substitutions and by backbone movements in loops surrounding the active site. The observed changes in the mutant structures are partly governed by coupled mutations, some of which are necessary to remove steric clashes or to allow backbone movements to occur. The importance of interactions between substitutions suggests that efficient directed evolution strategies should allow for compensating and synergistic mutations during library design.


  • Organizational Affiliation

    Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Halohydrin dehalogenase
A, B
254Agrobacterium tumefaciensMutation(s): 37 
Gene Names: hheC
EC: 4.5.1
UniProt
Find proteins for Q93D82 (Rhizobium radiobacter)
Explore Q93D82 
Go to UniProtKB:  Q93D82
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ93D82
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
1H1
Query on 1H1

Download Ideal Coordinates CCD File 
C [auth A]ethyl (3R)-4-cyano-3-hydroxybutanoate
C7 H11 N O3
LOQFROBMBSKWQY-ZCFIWIBFSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
D [auth B]CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.49 Å
  • R-Value Free: 0.249 
  • R-Value Work: 0.199 
  • R-Value Observed: 0.202 
  • Space Group: P 43 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 104.682α = 90
b = 104.682β = 90
c = 121.191γ = 90
Software Package:
Software NamePurpose
ADSCdata collection
PHASESphasing
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2013-02-20
    Type: Initial release
  • Version 1.1: 2013-05-15
    Changes: Database references
  • Version 1.2: 2018-01-24
    Changes: Structure summary
  • Version 1.3: 2023-09-20
    Changes: Data collection, Database references, Derived calculations, Refinement description