Elucidating the Role of O2 Uncoupling for the Adaptation of Bacterial Biodegradation Reactions Catalyzed by Rieske Oxygenases

Oxygenation of aromatic and aliphatic hydrocarbons by Rieske oxygenases is the initial step of various biodegradation pathways for environmental organic contaminants. Microorganisms carrying Rieske oxygenases are able to quickly adapt their substrate spectrum to alternative carbon and energy sources that are structurally related to the original target substrate, yet the molecular events responsible for this rapid adaptation are not well understood. Here, we evaluated the hypothesis that reactive oxygen species (ROS) generated by unproductive activation of O2, the so-called O2 uncoupling, in the presence of the alternative substrate exert a selective pressure for increasing the oxygenation efficiency of Rieske oxygenases. To that end, we studied wild-type 2-nitrotoluene dioxygenase (2NTDO) from Acidovorax sp. strain JS42 and five enzymes variants that have evolved from laboratory evolution experiments with 3- and 4-nitrotoluene as alternative growth substrates. The enzyme variants showed a substantially increased oxygenation efficiency towards the new target substrates concomitant with a reduction of ROS production, while mechanisms and kinetics of enzymatic O2 activation remained unchanged. Structural analyses and docking studies suggest that point mutations in enzyme variants occurred at residues lining both substrate and O2 transport tunnels enabling tighter binding of the target substrates in the active site. Increased oxygenation efficiencies measured in vitro for the various enzyme (variant)-substrate combinations correlated linearly with in vivo changes in growth rates for evolved Acidovorax strains expressing the variants. Our data suggest that the selective pressure from oxidative stress towards more efficient oxygenation by Rieske oxygenases was most notable when O2 uncoupling exceeded 60%.