Expression and Purification of Three Dioxygenases from the Plant Arabidopsis thaliana to Determine Their Possible Role in Phenanthrene Detoxification

Polycyclic aromatic hydrocarbons (PAHs) pollution is a life-threatening problem affecting humans because of its carcinogenic and mutagenic properties. PAHs consist of two or more benzene rings fused together, and are a class of hydrophobic toxins that emerge from the partial combustion of carbon-based fuels. They are also highly stable and persistent in the environment. Experiments with soil bacteria such as Pseudomonas indicate that microbes can be utilized in the bioremediation of these pollutants. Bacterial dioxygenases (DOXs) are enzymes capable to oxidize PAHs by transferring oxygen from O₂, de-stabilizing the chemical ring structure of PAHs. The purpose of this research is to ascertain if plant DOXs have the capabilities like bacterial DOXs to modify PAHs. There are fifty-two DOXs in the plant model system Arabidopsis thaliana. Six Arabidopsis DOX mRNAs are over-expressed when plants are exposed to phenanthrene, a three-ring PAH. Three of those six DOX genes (flavonol synthase, flavonone 3-hydroxylase and 2-oxoglutarate-dependent dioxygenase) were cloned and expressed in Eschirichia coli. The enzymes were purified from cell extracts and in vitro enzymatic assays were done to determine whether modifications to phenanthrene occurs. Using a scanning fluorometer to monitor changes in the ring structure, preliminary fluorescent emission results suggest that these purified enzymes are modifying PAHs, indicating that plant DOXs can be used in detoxification and phytoremediation strategies.