4AO9

Biochemical properties and crystal structure of a novel beta- phenylalanine aminotransferase from Variovorax paradoxus


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.182 
  • R-Value Work: 0.167 
  • R-Value Observed: 0.168 

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This is version 1.3 of the entry. See complete history


Literature

Biochemical Properties and Crystal Structure of a Novel Beta-Phenylalanine Aminotransferase from Variovorax Paradoxus

Crismaru, C.G.Wybenga, G.G.Szymanski, W.Wijma, H.J.Wu, B.Dewildeman, S.Poelarends, G.J.Dijkstra, B.W.Janssen, D.B.

(2013) Appl Environ Microbiol 79: 185

  • DOI: https://doi.org/10.1128/AEM.02525-12
  • Primary Citation of Related Structures:  
    4AO9, 4AOA

  • PubMed Abstract: 

    By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-β-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.


  • Organizational Affiliation

    Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
BETA-PHENYLALANINE AMINOTRANSFERASE
A, B
454Variovorax paradoxusMutation(s): 0 
UniProt
Find proteins for H8WR05 (Variovorax paradoxus)
Explore H8WR05 
Go to UniProtKB:  H8WR05
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupH8WR05
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.182 
  • R-Value Work: 0.167 
  • R-Value Observed: 0.168 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 88.74α = 90
b = 100.11β = 90
c = 104.799γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
XDSdata reduction
SCALAdata scaling
PHASERphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-10-24
    Type: Initial release
  • Version 1.1: 2012-11-07
    Changes: Database references
  • Version 1.2: 2012-11-14
    Changes: Other
  • Version 1.3: 2013-01-16
    Changes: Database references