Environmental microorganisms capable of oxidative biodegradation have been shown to evolve their enzymatic machinery upon exposure to xenobiotic compounds. Their biodegradation makes anthropogenic contaminants available as alternative sources of carbon and energy To that end, adaptation processes have been postulated that would be initiated by unproductive O 2 activation concomitant with production of ROS during the first chemical steps of contaminant transformation. Many biochemical processes are indeed necessary to lead from O 2 uncoupling, ROS release, and ROS scavenging to beneficial mutations that ultimately allow for expression of oxygenases with improved function. Yet, our work suggests that the changes of the efficiency of substrate oxygenation could would hint at the occurrence of this complex adaptation process. Point mutations at the terminal oxygenase of 2NTDO acquired from two independent laboratory evolution experiments both led to variants that enabled evolved strains to grow on alternative substrates concomitant with a substantial efficiency improvement of the oxygenases towards these alternative substrates. Analysis of sequence data also confirms the relevance of these variants in an environmental context through the presence of all variants in environmental sequences with relatively high natural abundance and taxonomic distributions among 2NTDO homologues (Figure ).