Structural and mechanistic insights into dioxin dioxygenase (DxnA1A2) from Rhizorhabdus wittichii RW1: A key enzyme for biodegradation of hazardous dioxins and furans.

Dioxin Dioxygenase (DxnA1A2) is one of the unique enzymes that can catalyze the angular dioxygenation of dibenzo-p-dioxin (DD) and dibenzofuran (DF). This enzyme has the potential to degrade other polychlorinated persistent organic compounds as well, which pose a serious threat to the environment and human health. In this study, the crystal structure of the dioxin dioxygenase was determined for the first time from Rhizorhabdus wittichii RW1. DxnA1A2 from RW1 is an effective enzyme against these substrates. The crystal structure provided critical insights into the α3β3 heterohexamer organization of the enzyme, coordination of mononuclear iron, and iron-sulphur complex. The enzyme was biochemically characterized by Oxygraph, and the product formation was validated by liquid chromatography-mass spectrometry. Biophysical characterization by circular dichroism and Differential Scanning Calorimetry indicated its mesophilic pH and temperature tolerance. Docking and Molecular dynamics simulation results elucidated the binding modes of the substrates, the critical catalytic residues, and the active site architecture. Based on structural comparisons, various strategies for mutagenesis have been proposed that can further enhance DxnA1A2 into an effective enzyme for the bioremediation of dioxins, furans, and related compounds. The efficiency of this enzyme in terms of yield, kinetics, stability in solution, the crystal structure, theoretical computations, biophysical and biochemical assays, paves the path for its scale-up and utilization for designing sustainable bioremediation strategies.