Computational Interpretation of Functional Divergence of VOC Family Catechol Dioxygenases in Bacillus thuringiensis HHY919: Insights from Homology Modeling and Molecular Docking

Abstract

Catechol 2,3-dioxygenase (C23O) is the rate-limiting enzyme in the meta-cleavage pathway of aromatic compound degradation, yet its functional annotation within the structurally conserved VOC superfamily remains challenging due to high sequence homology. Here, we isolated a catechol-degrading strain from bovine feces and identified it as Bacillus thuringiensis HHY919 via whole-genome sequencing. Genome annotation revealed four VOC genes sharing the same COG annotation (“catechol 2,3-dioxygenase”) but divergent KO annotations (two as glyoxalases and two as C23O), suggesting functional divergence. Using homology modeling and molecular docking, we compared their binding affinities toward catechol and 11 derivatives. All four proteins showed typical meta-cleavage binding energies (−4.9 to −5.4 kcal/mol), with slightly more favorable binding than an ortho-cleavage control. Notably, gene4224 exhibited the broadest and strongest predicted affinities in virtual screening against 212 compounds, particularly for trichlorophenol (−6.0 kcal/mol) and complex natural products (qvina_score ≤ −7.6 kcal/mol). Phylogenetic analysis and docking results jointly identified gene3355 and gene4224 as computationally prioritized C23O candidates, with gene4224 recommended as the top candidate for future enzyme engineering and bioremediation studies. This study provides a computational workflow for resolving functional ambiguity in VOC family enzymes and generating testable hypotheses for experimental validation.