title: 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.
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 α4β4 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 Fe(2+) increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage 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(-1)sec(-1), and for 2,2',3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10(-2) μM(-1)sec(-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.
authors.affiliation: a Department of Bioscience and Engineering , Shibaura Institute of Technology , Saitama , Japan.|b Biotechnology Research Center , The University of Tokyo , Tokyo , Japan.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Kotake
authors.affiliation: b Biotechnology Research Center , The University of Tokyo , Tokyo , Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Matsuzawa
authors.affiliation: b Biotechnology Research Center , The University of Tokyo , Tokyo , Japan.
authors.forename: Chiho
authors.initials: C
authors.lastname: Suzuki-Minakuchi
authors.affiliation: b Biotechnology Research Center , The University of Tokyo , Tokyo , Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Okada
authors.affiliation: b Biotechnology Research Center , The University of Tokyo , Tokyo , Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nojiri
authors.affiliation: a Department of Bioscience and Engineering , Shibaura Institute of Technology , Saitama , Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Iwata
mesh_terms: D001673:Biodegradation, Environmental; D005449:Fluorenes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005449:Fluorenes; D011994:Recombinant Proteins; C041509:fluorene; D010105:Oxygenases; C439587:extradiol dioxygenase
keywords: Rhodococcus; bacteria; characterization; extradiol dioxygenase; fluorene