| Summary: | Polycyclic aromatic hydrocarbons (PAHs) are widespread hazardous, hydrophobic pollutants made of fused benzene rings. Besides of being present in large quantities in petroleum and related products they are formed generally during the incomplete combustion of organic matter. During evolution, bacteria have become capable of using such recalcitrant compounds as growth substrates. Hundreds of species of bacteria have been identified that can metabolise a wide range of contaminants, being of potential use in bioremediation strategies. The types of molecules that can be degraded depend on the metabolic pathway in each species.
The genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The a and b subunits of a dioxygenase complex (FlnA1A2), showing 63% and 51% sequence identity respectively, with the subunits of an angular dioxygenase from the Gram-positive dibenzofuran degrader Terrabacter sp. strain 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. strain DBF63. Quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1A2.
Sphingomonas sp. strain LH128 harbors a naphthalene dioxygenase (PhnA1A2f) responsible for the oxidation of PAHs made of up to four rings, monochlorinated biphenyls and dibenzo-p-dioxin. PhnA1A2f shows exceptionally broad substrate specificity towards various pollutants in contrast to other naphthalene dioxygenases whose activity is limited to two- and three-ring PAHs. Moreover a conserved catabolic gene cluster could be isolated harboring 13 genes involved in PAH degradation. Sequence comparison with the initial dioxygenase of Sphingomonas sp. strain CHY-1 showed that the amino acids lining the catalytic pocket are conserved. The variation in substrate specificity of the two dioxygenases demonstrates that substitutions outside of the catalytic pocket can have marked effects on the substrate range of dioxygenase enzymes.
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