Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase, in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Tatsuro Kotake, Jun Matsuzawa, Chiho Suzuki-Minakuchi, Kazunori Okada, Hideaki Nojiri, Kenichi Iwata

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an a4β4 heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe2+ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed metacleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The Km and kcat/Km values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10-2 μM-1sec-1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10-2 μM-1sec-1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.

Original languageEnglish
Pages (from-to)719-725
Number of pages7
JournalBioscience, Biotechnology and Biochemistry
Volume80
Issue number4
DOIs
Publication statusPublished - 2016

Keywords

  • Bacteria
  • Characterization
  • Extradiol dioxygenase
  • Fluorene
  • Rhodococcus

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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