1SCK

K236L mutant of hydroxynitrile lyase from Hevea brasiliensis in complex with acetone


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.194 
  • R-Value Work: 0.170 
  • R-Value Observed: 0.170 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Reaction mechanism of hydroxynitrile lyases of the alpha/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236

Gruber, K.Gartler, G.Krammer, B.Schwab, H.Kratky, C.

(2004) J Biol Chem 279: 20501-20510

  • DOI: https://doi.org/10.1074/jbc.M401575200
  • Primary Citation of Related Structures:  
    1SC9, 1SCI, 1SCK, 1SCQ

  • PubMed Abstract: 

    The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.


  • Organizational Affiliation

    Institut für Chemie, Physikalische Chemie, Karl-Franzens Universitaät Heinrichstrasse 28, A-8010 Graz, Austria.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
(S)-acetone-cyanohydrin lyase257Hevea brasiliensisMutation(s): 1 
Gene Names: HNL
EC: 4.1.2.39
UniProt
Find proteins for P52704 (Hevea brasiliensis)
Explore P52704 
Go to UniProtKB:  P52704
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP52704
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.194 
  • R-Value Work: 0.170 
  • R-Value Observed: 0.170 
  • Space Group: C 2 2 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 47.516α = 90
b = 106.936β = 90
c = 128.116γ = 90
Software Package:
Software NamePurpose
CNSrefinement
MAR345data collection
SCALEPACKdata scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-06-29
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Refinement description
  • Version 1.4: 2021-11-10
    Changes: Data collection, Database references, Derived calculations
  • Version 1.5: 2023-10-25
    Changes: Data collection, Refinement description