The management and mitigation of chemical pollution are key elements of sustainable development initiatives that aim to provide safe and clean water. While environmental scientists are developing the capabilities to assess the fate, (eco)toxicity and risks of a plethora of synthetic chemicals comprehensively, notorious pollution scenarios and decontamination challenges call for targeted and case-specific evaluation of chemical hazards. Here we review the utility and perspectives of compound-specific isotope analysis for obtaining an understanding of environmental processes that allows one to identify pollution sources, assess contaminant (bio)transformation and gain insights into reaction pathways. Using three prototypical scenarios of water contamination, namely point-source pollution of groundwater at contaminated sites, diffuse pollution of soils and surface waters through pesticide use and the abatement of pharmaceuticals and disinfection by-products in water treatment systems, we illustrate both success stories of compound-specific isotope analysis and current developments to address challenges for future applications.

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 spectra 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 on the bacterium for increasing the oxygenation efficiency of Rieske oxygenases. To that end, we studied wild-type 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 and five enzyme variants that have evolved from adaptive laboratory evolution experiments with 3- and 4-nitrotoluene as alternative growth substrates. The enzyme variants showed a substantially increased oxygenation efficiency toward 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 amino acid substitutions 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 toward more efficient oxygenation by Rieske oxygenases was most notable when O₂ uncoupling exceeded 60%.

Rieske oxygenases are known as catalysts that enable the cleavage of aromatic and aliphatic C–H bonds in structurally diverse biomolecules and recalcitrant organic environmental pollutants through substrate oxygenations and oxidative heteroatom dealkylations. Yet, the unproductive O₂ activation, which is concomitant with the release of reactive oxygen species (ROS), is typically not taken into account when characterizing Rieske oxygenase function. Even if considered an undesired side reaction, this O₂ uncoupling allows for studying active site perturbations, enzyme mechanisms, and how enzymes evolve as environmental microorganisms adapt their substrates to alternative carbon and energy sources. Here, we report on complementary methods for quantifying O₂ uncoupling based on mass balance or kinetic approaches that relate successful oxygenations to total O₂ activation and ROS formation. These approaches are exemplified with data for two nitroarene dioxygenases (nitrobenzene and 2-nitrotoluene dioxygenase) which have been shown to mono- and dioxygenate substituted nitroaromatic compounds to substituted nitrobenzylalcohols and catechols, respectively.