Elucidation of the biochemical factors governing the enzymatic desulfonation of 6:2 fluorotelomer sulfonate: Purification and enzymatic characterization of Escherichia and Gordonia monooxygenases

Fluorotelomer sulfonate degradation is thought to be a rate limiting step in the degradation of fluorinated surfactants in activated sludge wastewater treatment. Aliphatic sulfonates are structurally similar to fluorotelomer sulfonates and are partially degraded by alkanesulfonate monooxygenases. To understand the metabolism of 6:2 fluorotelomer sulfonate (6:2 FTSA), two nitrilotriacetate monooxygenases (ISGA 1218 and 1222) from Gordonia NB4-1Y, along with the Escherichia coli alkanesulfonate monooxygenase (SsuD) were cloned into the pMAL-c2 and pET28b protein production vectors, purified, given the E. coli flavin reductase (Fre) as a source of reduced flavin production and challenged in vitro with octane sulfonate and 6:2 FTSA. A combination of gas chromatography, mass spectrometry and spectrophotometry revealed that ISGA 1218 and 1222 were inactive against octane sulfonate and 6:2 FTSA; SsuD, however, was active against both octane sulfonate and 6:2 FTSA, removing up to 120 μM of added octane sulfonate and up to 130 μM 6:2 FTSA during a two hour reaction at room temperature. These data are preliminary, however, suggest sulfite is produced as a product of 6:2 FTSA degradation and likely represents the first evidence of the biochemical transformation of fluorinated surfactants by a purified bacterial enzyme. Further, Escherichia coli BL21(DE3) was found to tentatively grow on 6:2 FTSA prepared in water as a sole source of sulfur reaching OD660 of 0.32 as compared to 0.15 of the no sulfur treatment after 48 hours. Attempts to produce Gordonia NB4-1Y mutants via conjugation or electroporation with pK18mobsacB1218A or pK18mobsacB1222AB were unsuccessful. Transformation of 6:2 FTSA by SsuD suggests the ssu operon in E. coli is responsible for the desulfonation of fluorotelomer sulfonates and presents the ssuABC system as a potentially versatile model to study the in vivo import of fluorotelomer sulfonates. Furthermore, assignment of 6:2 FTSA degradation to the ssu operon suggests that fluorotelomer sulfonate degradation may be enriched under sulfur starvation conditions. Identifying the enzymes responsible for aliphatic sulfonate degradation in Gordonia NB4-1Y is paramount to understanding the metabolism of fluorinated surfactants by this bacterium. Here we present one luciferase-like class flavin-dependent oxidoreductase (ISGA 08960) as candidate for further biochemical analysis.