Structural and mechanistic insights into Edo4, an extradiol dioxygenase in dioxin degradation

Extradiol dioxygenases (EDOs) are integral ring-cleaving enzymes that bridge the catabolic pathways between dioxygenase and hydrolase enzymes. However, the structural basis for substrate recognition and active site access in EDOs of the dioxin degradation pathway remains unclear. Here, we report the crystal structures of Edo4_RW1 from Rhizorhabdus wittichii RW1 in the apo form and in complex with the substrate analogue 2-hydroxybiphenyl (2-HBP) at 2.0 and 2.8 Å resolution, respectively. Structural insights helped establish its canonical Type I extradiol fold, featuring the conserved mononuclear iron, Fe(II) binding triad and substrate-binding pocket, but with a notably divergent C-terminus and β-hairpin region that partitions the C-domain funnel into two entry channels. Docking and MD simulations into this experimentally determined active-site pocket further unravel the mechanistic aspects of the plausible bidentate binding modes adopted by both substrates and their analogues, while they exploit different subsites of the hydrophobic distal pocket. The structural data, docking, and MD simulations, and comparative analyses suggest a working model in which Edo4_RW1 has evolved a more elaborate, dynamically gated access architecture than classical single-funnel extradiol dioxygenases in order to handle bulky, asymmetrically substituted 2,2',3-THB and THDE intermediates. These findings provide mechanistic insights into substrate recognition and access in Edo4_RW1 and reveal how structural adaptations expand the catalytic scope of extradiol dioxygenases.