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


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.


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.



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


  1. Armengaud J, Timmis KN (1997) Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. strain RW1. Eur J Biochem 247:833–842CrossRefGoogle Scholar
  2. Armengaud J, Timmis KN (1998) The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp. strain RW1 is related to class-I cytochrome P450-type reductases. Eur J Biochem 253:437–444CrossRefGoogle Scholar
  3. Armengaud J, Happe B, Timmis KN (1998) Genetic analysis of dioxin dioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersed on the genome. J Bacteriol 180:3954–3966PubMedGoogle Scholar
  4. Armengaud J, Gaillard J, Timmis KN (2000) A second [2Fe-2S] ferredoxin from Sphingomonas sp. strain RW1 can function as an electron donor for the dioxin dioxygenase. J Bacteriol 182:2238–2244CrossRefGoogle Scholar
  5. Bünz PV, Cook AM (1993) Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system. J Bacteriol 175:6467–6475Google Scholar
  6. Eaton RW (2001) Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B. J Bacteriol 183:3689–3703CrossRefGoogle Scholar
  7. Goodfellow BJ, Macedo AL, Rodrigues P, Moura I, Wray V, Moura JJG (1999) The solution structure of a [3Fe-4S] ferredoxin: oxidized ferredoxin II from Desulfovibrio gigas. J Biol Inorg Chem 4:421–430CrossRefGoogle Scholar
  8. Gorst CM, Yeh Y-H, Teng Q, Calzolai L, Zhou Z-H, Adams MWW, Mar GNL (1995) 1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure. Biochemistry 34:600–610Google Scholar
  9. Habe H, Omori T (2002) Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria. Biosci Biotechnol Biochem 67:225–243CrossRefGoogle Scholar
  10. Habe H, Chung J-S, Lee J-H, Kasuga K, Yoshida T, Nojiri H, Omori T (2001) Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenase with different features. Appl Environ Microbiol 67:3610–3617CrossRefPubMedGoogle Scholar
  11. Habe H, Miyakoshi M, Chung J-S, Kasuga K, Yoshida T, Nojiri H, Omori T (2003) Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in Terrabacter sp. strain DBF63. Appl Microbiol Biotechnol 61:44–54Google Scholar
  12. Habe H, Chung J-S, Kato H, Ayabe Y, Kasuga K, Yoshida T, Nojiri H, Yamane H, Omori T (2004) Characterization of the upper pathway genes for fluorene metabolism in Terrabacter sp. strain DBF63. J Bacteriol 186:5938–5944CrossRefGoogle Scholar
  13. Iida T, Mukouzaka Y, Nakamura K, Kudo T (2002) Plasmid-borne genes code for an angular dioxygenase involved in dibenzofuran degradation by Terrabacter sp. strain YK3. Appl Environ Microbiol 61:3716–3723CrossRefGoogle Scholar
  14. Inoue K, Widada J, Nakai S, Endoh T, Urata M, Ashikawa Y, Shintani M, Saiki Y, Yoshida T, Habe H, Omori T, Nojiri H (2004) Divergent structures of carbazole degradative car operons isolated from Gram-negative bacteria. Biosci Biotechnol Biochem 68:1467–1480CrossRefPubMedGoogle Scholar
  15. Kasuga K, Habe H, Chung J-S, Yoshida T, Nojiri H, Yamane H, Omori T (2001) Isolation and characterization of the genes encoding a novel oxygenase component of angular dioxygenase from the Gram-positive dibenzofuran-degrader Terrabacter sp. strain DBF63. Biochem Biophys Res Commun 283:105–204CrossRefGoogle Scholar
  16. Kilbane II JJ, Daram A, Abbasian J, Kayser KJ (2002) Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum. Biochem Biophys Res Commun 297:242–248CrossRefGoogle Scholar
  17. Martin VJJ, Mohn WW (1999) A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9. J Bacteriol 181:2675–2682Google Scholar
  18. Nam J-W, Nojiri H, Noguchi H, Uchimura H, Yoshida T, Habe H, Yamane H, Omori T (2002) Purification and characterization of carbazole 1,9a-dioxygenase, a three-component dioxygenase system of Pseudomonas resinovorans strain CA10. Appl Environ Microbiol 68:5882–5890CrossRefPubMedGoogle Scholar
  19. Nojiri H, Omori T (2002) Molecular bases of aerobic bacterial degradation of dioxins: involvement of angular dioxygenation. Biosci Biotechnol Biochem 66:2001–2016CrossRefPubMedGoogle Scholar
  20. Nojiri H, Nam J-W, Kosaka M, Morii K, Takemura T, Furihata K, Yamane H, Omori T (1999) Diverse oxygenation catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp. strain CA10. J Bacteriol 181:3105–3113Google Scholar
  21. Nojiri H, Kamakura M, Urata M, Tanaka T, Chung J-S, Takemura T, Yoshida T, Habe H, Omori T (2002) Dioxin catabolic genes are dispersed on the Terrabacter sp. DBF63 genome. Biochem Biophys Res Commun 296:233–240CrossRefGoogle Scholar
  22. Noumura T, Habe H, Widada J, Chung J-S, Yoshida T, Nojiri H, Omori T (2004) Genetic characterization of the dibenzofuran-degrading Actinobacteria carrying dbfA1A2 gene homologues isolated from activated sludge. FEMS Microbiol Lett 239:147–155CrossRefGoogle Scholar
  23. O’Keefe DP, Gibson KJ, Emptage MH, Lenstra R, Romesser JA, Litle PJ, Omer CA (1991) Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus. Biochemistry 30:447–455Google Scholar
  24. Saito A, Iwabuchi T, Harayama S (2000) A novel phenanthrene dioxygenase from Nocardioides sp. strain KP7: expression in Escherichia coli. J Bacteriol 182:2134–2141CrossRefGoogle Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  26. Sato S, Nam J-W, Kasuga K, Nojiri H, Yamane H, Omori T (1997) Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10. J Bacteriol 179:4850–4858PubMedGoogle Scholar
  27. Takagi T, Nojiri H, Yoshida T, Habe H, Omori T (2002) Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans. Biotechnol Lett 24:2099–2106CrossRefGoogle Scholar
  28. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific-gap penalties and weight matrix choice. Nucleic Acid Res 22:4673–4680PubMedGoogle Scholar

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

Personalised recommendations