Insights into the binding interaction of substrate with catechol 2,3-dioxygenase from biophysics point of view

Abstract

This study aims to clarify the interaction mechanism of substrate with catechol 2,3-dioxygenase (C23O) through multi-technique combination. A novel C23O (named C23O-2G) was cloned, heterogeneously expressed, and identified as a new member in subfamily I.2 of extradiol dioxygenases. Based on the simulations of molecular docking and dynamics, the exact binding sites of catechol on C23O-2G were identified, and the catalytic mechanism mediated by key residues was proposed. The roles of the predicted residues during catalysis were confirmed by site-directed mutagenesis, and the mutation of Thr254 could significantly increase catalytic efficiency and substrate specificity of C23O-2G. The binding and thermodynamic parameters obtained from fluorescence spectra suggested that catechol could effectively quench the intrinsic fluorescence of C23O-2G via static and dynamic quenching mechanisms and spontaneously formed C23O-2G/catechol complex by the binding forces of hydrogen bond and van der Waals force. The results of UV-vis spectra, synchronous fluorescence, and CD spectra revealed obvious changes in the microenvironment and conformation of C23O-2G, especially for the secondary structure. The atomic force microscope images further demonstrated the changes from an appearance point of view. This study could improve our mechanistic understanding of representative dioxygenases involved in aromatic compound degradation.

Keywords: Aromatic ring cleavage; Binding interaction; Catalytic mechanism; Catechol 2,3-dioxygenase; Multi-spectroscopy.