Substrate specificities of aromatic ring-hydroxylating oxygenases of an uncultured gammaproteobacterium from chronically-polluted subantarctic sediments

Aromatic ring-hydroxylating oxygenases (RHOs) are multicomponent enzymes that catalyze the vicinal hydroxylation of aromatic rings to produce cis-dihydrodiols, a key step in the aerobic biodegradation of aromatic compounds. In this work, we describe the characterization of three RHOs of an uncultured gammaproteobacterium from chronically polluted Subantarctic intertidal sediments. Sequences encoding the α and β subunits of these RHOs, classified as class A type III, and one set of the corresponding electron transfer partners, were identified in a 34 Kb fragment from a metagenomic fosmid library. Structural modeling and docking analyses suggested that the active sites of these enzymes accommodated different set of substrates. The three enzymes, including the electron transfer components, were expressed in Escherichia coli and purified. The enzyme with the largest predicted catalytic pocket and wider diameter channels presented remarkably relaxed substrate specificity, including 2–4 ring PAHs (phenanthrene, pyrene, fluoranthene and naphthalene). The other two RHOs were stricter in their substrate specificity, and hydroxylated biphenyl and naphthalene more efficiently. These results suggest the evolution of compatible RHO enzymes within a single catabolic gene cluster in this microorganism. This work increases our understanding of the PAH-degrading capabilities of uncultured bacteria from cold coastal environments. [Display omitted] •Three aromatic ring-hydroxylating oxygenases were identified in a metagenomic library.•Differences in size and shape of active sites were predicted by molecular modeling.•The oxygenases and essential electron donors ferredoxin and reductase were purified.•Distinct catalytic efficiencies were confirmed for different aromatic substrates.•Oxygenases with different substrate range provide expansion in catabolic capabilities.