Anthropogenic organic contaminants in natural and engineered environments e.g. (waste-) water treatment plants are a risk for food and water safety. Microorganisms can quickly adapt their enzymatic machinery to degrade these compounds. In various pathways degradation of anthropogenic compounds is initiated by oxygenation catalyzed by Rieske oxygenases. The oxygenated products are then used as alternative sources of carbon and energy in standard catabolic and biosynthetic pathways.

Rieske non-heme ferrous iron oxygenases are an important class of enzymes in this respect due to their ability to accept many substrates that reflect the spectrum of man-made chemicals such as industrial chemicals, pharmaceuticals, or pesticides. The study now establishes a link between microbial adaptation to novel substrates, changes within the enzyme protein sequence/ structure and the oxygenation efficacy of the oxygenases involved.

The work focuses on a Riske oxygenase studied previously: 2-nitrotoluene dioxygenase (2NTDO) from Acidovorax sp. Strains expressing the enzyme can adapt from 2- nitrotoluene to 3- and 4- nitrotoluene once exposed to these alternative substrates for a couple of weeks. Enzyme variants isolated from the adapted strains contained one or two amino acid substitutions. Five of these variants were used in the present study to evaluate the hypothesis that in the presence of alternative substrates reactive oxygen species (ROS) are generated by un-productive activation of O₂, the so-called O₂ uncoupling, which puts a selective pressure on the bacterium to increase the oxygenation efficiency of Rieske oxygenases.

Comparing the oxygenation efficiency of the wild-type enzyme and the variants using 4-nitrotoluene as a substrate, the mutants used the activated O₂ more effectively than the wild-type enzyme.

Wild-type 2NTDO and its variants were then tested towards four nitrobenzene substrates and three nitrotoluene isomers. The single amino acid substitution M248I was the prerequisite for the gradual adaptation to 4-nitrotoluene in the 2-NTDO variants. Oxygenation efficiency increased from 6% (wild- type) to 57% (double mutant). A similar trend was seen for variants isolated from strains growing on 3-nitrotoluene. In both cases the effect was specific to the substrate the respective strains had been grown on. Most other substrates were even oxygenated less efficiently.

Increased oxygenation efficiencies of the various enzyme/variant – substrate combinations measured in-vitro correlated well with changes in growth rates and NO₂^- production rates for the evolved Acidovorax spec. strains expressing the respective enzyme variants. 

Amino acid changes within the enzyme structure were analyzed using CaverDock. The model suggests that the mutated residues in the variants isolated from 4-NT selection were located within the O₂ transport tunnel of the WT enzyme, while mutations identified from variants from the 3NT selection were at amino acids located in the substrate binding tunnel. Tighter binding of the target substrates within the active site correlated with decreased O₂-uncoupling and ROS production.

The data suggest that the selective pressure from oxidative stress towards more efficient oxygenation by Riske oxygenases was most notable when O₂ uncoupling was above 60%. Thus, oxidative stress from O₂ uncoupling is not the sole selecting factor once the value is below 60%. Below this threshold other factors than oxygenation become beneficial and determine fitness.

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

C. E. Bopp C. E., Bernet N. M., Meyer F., Khan R., Robinson S. L., Kohler H.-P. E., Buller R.  & Hofstetter T. B. (2024). ACS ENVIRONMENTAL AU (2024) May 14.

https://doi.org/10.1021/acsenvironau.4c00016

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