Applied Microbiology and Biotechnology

, Volume 91, Issue 4, pp 1049–1060

Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1

  • Martin Hesseler
  • Xenia Bogdanović
  • Aurelio Hidalgo
  • Jose Berenguer
  • Gottfried J. Palm
  • Winfried Hinrichs
  • Uwe T. Bornscheuer
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-011-3328-x

Cite this article as:
Hesseler, M., Bogdanović, X., Hidalgo, A. et al. Appl Microbiol Biotechnol (2011) 91: 1049. doi:10.1007/s00253-011-3328-x

Abstract

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/β-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The Km and kcat values for 1-bromobutane were 24.0 mM and 8.08 s−1. Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.

Keywords

Haloalkane dehalogenase Plesiocystis pacifica Biochemical characterization Structure 

Supplementary material

253_2011_3328_MOESM1_ESM.pdf (123 kb)
ESM1 (PDF 122 kb)

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Martin Hesseler
    • 1
    • 3
  • Xenia Bogdanović
    • 2
  • Aurelio Hidalgo
    • 3
  • Jose Berenguer
    • 3
  • Gottfried J. Palm
    • 2
  • Winfried Hinrichs
    • 2
  • Uwe T. Bornscheuer
    • 1
  1. 1.Department of Biotechnology and Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany
  2. 2.Department of Molecular Structural Biology, Institute of BiochemistryGreifswald UniversityGreifswaldGermany
  3. 3.Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (UAM-CSIC)Universidad Autónoma de MadridMadridSpain

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