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 Fe²⁺ 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 K_m and k_cat/K_m 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.

Graphical abstract

Type II extradiol dioxygenases (EDOs) are classified into 5 subgroups, and FlnD1D2 constitutes the distinct group from previously investigated EDOs.

Key words:

• bacteria
• characterization
• extradiol dioxygenase
• fluorene
• Rhodococcus

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

Abbreviations: Ap, ampicillin; DF, dibenzofuran; FN, fluorene; km, kanamycin; LB, Luria–Bertani; PAHs, polycyclic aromatic hydrocarbons; SD, Shine–Dalgarno; 2,2′,3-THBP, 2,2′,3-trihydroxybipenyl, 2,3-DHBP, 2,3-dihydroxybiphenyl; 2′-carboxy-2,3-DHBP, 2′-carboxy-2,3-dihydroxybiphenyl.