In silico and in vitro exploration of a tyrosinase for biocatalytic production of catechols

Tyrosinases are binuclear copper enzymes widely used for the hydroxylation of phenolic compounds, yet most exhibit higher activity towards diphenols than monophenols, limiting their utility for catechol synthesis. However, a tyrosinase from Ralstonia pseudosolanacearum GMI1000 (RsTyr) is unusual in favouring monophenol substrates, making it a promising biocatalyst. Here, we combined molecular docking with kinetic characterisation to explore RsTyr's substrate range across eleven monophenols of industrial relevance. Docking studies using homology and AlphaFold models identified key interactions between substrate side chains and catalytic residues, particularly N228, N232 and P239. Experimental validation confirmed broad substrate acceptance, with highest catalytic efficiency observed for resveratrol and tyrosol. Structural analysis suggests that terminal functional groups and sidechain branching strongly influence activity. These findings provide mechanistic insight into RsTyr's substrate specificity and inform strategies for biocatalytic production of valuable catechols.