1W1K

STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: Ile238Thr Mutant


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.55 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.207 
  • R-Value Observed: 0.210 

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This is version 1.3 of the entry. See complete history


Literature

Laboratory-Evolved Vanillyl-Alcohol Oxidase Produces Natural Vanillin

Van Den Heuvel, R.H.Van Den Berg, W.A.Rovida, S.Van Berkel, W.J.

(2004) J Biol Chem 279: 33492

  • DOI: https://doi.org/10.1074/jbc.M312968200
  • Primary Citation of Related Structures:  
    1W1J, 1W1K, 1W1L, 1W1M

  • PubMed Abstract: 

    The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.


  • Organizational Affiliation

    Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
VANILLYL-ALCOHOL OXIDASE
A, B
560Penicillium simplicissimumMutation(s): 1 
EC: 1.1.3.13
UniProt
Find proteins for P56216 (Penicillium simplicissimum)
Explore P56216 
Go to UniProtKB:  P56216
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP56216
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.55 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.207 
  • R-Value Observed: 0.210 
  • Space Group: I 4
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 129.896α = 90
b = 129.896β = 90
c = 133.383γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALAdata scaling
CCP4phasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-07-02
    Type: Initial release
  • Version 1.1: 2011-05-08
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2023-12-13
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description