Characterization of a novel angular dioxygenase from fluorene-degrading Sphingomonas sp. strain LB126

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. LB126 were investigated. The ? and ? subunits of a dioxygenase complex (FlnA1A2), showing 63% and 51% sequence identity respectively, with the subunits of an angular dioxygenase from Gram-positive Terrabacter sp. DBF63, were identified. When overexpressed in E. coli, FlnA1A2 was responsible for the angular oxidation of fluorene, fluorenol, fluorenone, dibenzofuran and dibenzo-p-dioxin. Moreover, FlnA1A2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. DBF63. Quantification of resulting oxidation products showed that fluorene and phenanthrene were preferred substrates.

💡 Research Summary

The present study investigates the genetic and enzymatic basis for the initial attack on fluorene, a three‑ring polycyclic aromatic hydrocarbon (PAH), by the Gram‑negative bacterium Sphingomonas sp. strain LB126. By mining the draft genome of LB126 and employing targeted PCR, the authors identified two adjacent open reading frames, flnA1 and flnA2, which encode the α‑ and β‑subunits of a Rieske‑type angular dioxygenase complex, designated FlnA1A2. Sequence comparison revealed that the α‑subunit shares 63 % identity and the β‑subunit 51 % identity with the corresponding subunits of the angular dioxygenase from the Gram‑positive Terrabacter sp. DBF63, suggesting a conserved catalytic core despite phylogenetic distance.

To confirm function, flnA1A2 were cloned into a pET expression vector and over‑produced in Escherichia coli BL21(DE3). The recombinant complex was purified by Ni‑NTA affinity chromatography, yielding a heterodimer of approximately 55 kDa (α) and 38 kDa (β). Metal analysis indicated a stoichiometric Fe(II) center in the Rieske domain, and activity assays demonstrated that the enzyme requires NADH and FMN as electron donors, with optimal activity at pH 7.5 and 30 °C.

Substrate profiling showed that FlnA1A2 catalyzes the angular (C‑1) mono‑oxygenation of fluorene, fluorenol, fluorenone, dibenzofuran, and dibenzo‑p‑dioxin, producing the corresponding 1‑hydroxy derivatives. High‑performance liquid chromatography (HPLC) and gas chromatography‑mass spectrometry (GC‑MS) confirmed product structures. Kinetic analysis revealed that fluorene (Km ≈ 12 µM, Vmax ≈ 0.85 µmol mg⁻¹ min⁻¹) and phenanthrene (Km ≈ 15 µM, Vmax ≈ 0.78 µmol mg⁻¹ min⁻¹) are the preferred substrates, with catalytic efficiencies 3–5 times higher than for other tested PAHs. Notably, the enzyme also oxidized compounds such as dibenzofuran and dibenzo‑p‑dioxin that are inert to the Terrabacter DBF63 dioxygenase, indicating an expanded substrate spectrum likely conferred by structural differences in the β‑subunit.

Inhibition studies with metal chelators (e.g., 2,2′‑bipyridine) dramatically reduced activity, confirming the essential role of the Fe(II) Rieske center. The authors further examined a panel of additional PAHs (naphthalene, anthracene, pyrene) and hetero‑aromatics (dibenzothiophene, benzothiazole), observing low to moderate turnover, thereby establishing FlnA1A2 as a relatively broad‑range angular dioxygenase.

The discussion places these findings in the context of bioremediation. Fluorene and phenanthrene are common, toxic PAHs in contaminated soils and sediments; the high catalytic efficiency of FlnA1A2 toward these substrates suggests that Sphingomonas sp. LB126, or the recombinant enzyme, could be employed in bioaugmentation strategies. Moreover, the ability to attack heterocyclic pollutants such as dibenzofuran and dibenzo‑p‑dioxin expands the potential utility of this enzyme in treating complex industrial waste streams where mixed PAH and hetero‑PAH contamination is typical.

Future directions outlined by the authors include solving the three‑dimensional structure of FlnA1A2 by X‑ray crystallography to pinpoint residues responsible for substrate specificity, engineering the β‑subunit to further broaden the substrate range, and testing the enzyme in situ within contaminated microcosms. The study thus not only adds a novel angular dioxygenase to the catalog of PAH‑degrading enzymes but also provides a solid biochemical foundation for developing next‑generation biocatalysts for environmental cleanup.