Kinetic isotope effects of C and N indicate different transformation mechanisms between atzA- and trzN-harboring strains in dechlorination of atrazine

Compound-specific stable isotope analysis provides an alternative method to insight into the biotransformation mechanisms of diffuse organic pollutants in the environment, e.g., the endocrine disruptor herbicide atrazine. Biotic hydrolysis process catalyzed by chlorohydrolase AtzA and TrzN plays an important role in the detoxification of atrazine, while the catalytic mechanism of AtzA is still speculative. To investigate the catalytic mechanism of AtzA and answer whether both enzymes catalyze hydrolytic dechlorination of atrazine by the same mechanism, in this study, apparent kinetic isotope effects (AKIE) for carbon and nitrogen were observed by three atzA-harboring bacterial isolates and their membrane-free extracts. The AKIEs obtained from atzA-harboring bacterial isolates (AKIE(C) = 1.021 +/- 0.010, AKIE(N) = 0.992 +/- 0.003) were statistically different from that of trzN-harboring strains (AKIE(C) = 1.040 +/- 0.006, AKIE(N) = 0.983 +/- 0.006), confirming the different activation mechanisms of atrazine preceding to nucleophilic aromatic substitution of Cl atom in actual enzymatic reaction catalyzed by AtzA and TrzN, despite the limitation of variable dual-element isotope plots. The lower degree of normal carbon and inverse nitrogen isotope fractionation observed from atzA-harboring strains, suggesting AtzA catalyzing hydrolytic dechlorination of atrazine by coordination of Cl and one aromatic N to the Fe2+ drawing electron density from carbon-chlorine bond that facilitating the nucleophilic attack, rather than in TrzN case that protonation of aromatic N increasing nucleophilic substitution of Cl atom. This study suggests considering the potential influences of phylogenetic diversity of bacterial isolates and evolution of enzymes on the applications of CSIA method in future study.

Automated summary

Compound-specific stable isotope analysis is an alternative method for investigating the biotransformation mechanisms of organic pollutants in the environment. This study investigated the catalytic mechanism of the herbicide atrazine by analyzing the apparent kinetic isotope effects (AKIE) of carbon and nitrogen. The results showed that the catalytic mechanisms of the enzymes AtzA and TrzN in the hydrolytic dechlorination of atrazine are different.