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Applied Microbiology and Biotechnology

, Volume 68, Issue 3, pp 336–345 | Cite as

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63

  • Terufumi Takagi
  • Hiroshi Habe
  • Takako Yoshida
  • Hisakazu Yamane
  • Toshio Omori
  • Hideaki NojiriEmail author
Biotechnologically Relevant Enzymes and Proteins

Abstract

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min−1 mg−1) and convert dibenzofuran to 2,2′,3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.

Keywords

Electron Paramagnetic Resonance Ferredoxin Reductase Extradiol Dioxygenase ORF16 Product CarAc 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgement

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Terufumi Takagi
    • 1
  • Hiroshi Habe
    • 1
  • Takako Yoshida
    • 1
  • Hisakazu Yamane
    • 1
  • Toshio Omori
    • 1
    • 2
  • Hideaki Nojiri
    • 1
    Email author
  1. 1.Biotechnology Research CenterThe University of TokyoTokyoJapan
  2. 2.Department of Industrial Chemistry, Faculty of EngineeringShibaura Institute of TechnologyTokyoJapan

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