Endosulfan is a hexachlorinated cyclodiene pesticide that has been widely used, until a ban in many countries and a phase-out in some countries took effect in 2012. Endosulfan is toxic, not only to insects but also to vertebrates. It has been linked to the emergence of certain diseases, such as cancer and congenital malformations, in heavily contaminated places. Furthermore, it is transported in clouds from heavily contaminated places around the world. Thus, endosulfan has been detected in areas where it has never been used. Endosulfan is partially biodegraded in the soil: the sulfite is easily oxidized to a sulfate, which can then be hydrolyzed to give the endosulfan diol. The diol can be oxidized further to the corresponding lactone. Therefore, the metabolites of endosulfan that accumulate (the diol and lactone) still have the six carbon-chlorine bonds intact. We have discovered mutants of cytochrome P450cam, a camphor hydroxylase from a strain of the soil bacterium Pseudomonas putida, that completely dechlorinates endosulfan diol. The mutants were generated through a random mutagenesis scheme known as Sequence Saturation Mutagenesis (SeSaM), followed by expression and selection on endosulfan. Interestingly, the wild-type enzyme can convert endosulfan to the sulfate and the diol to the dialdehyde, but it cannot dechlorinate. The mutant enzymes dechlorinate the bicyclic framework and convert it to an aromatic metabolite. Accumulation of insecticides and their structurally related metabolites in the environment can contribute to the emergence of resistance and cross-resistance. Therefore, the discovery of complete biodegradation routes to recalcitrant pesticides is an important step in the management of resistance and the development of more sustainable agricultural practices.

Biodegradation of endosulfan: Discovery of new cytochrome P450<sub>cam</sub> mutants that completely dechlorinate endosulfan diol

248th National Meeting (2014)

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Biodegradation of endosulfan: Discovery of new cytochrome P450cam mutants that completely dechlorinate endosulfan diol

Biodegradation of endosulfan: Discovery of new cytochrome P450_cam mutants that completely dechlorinate endosulfan diol