Functional characterization of a novel p-coumarate 3-hydroxylase from Trametes versicolor

Lignin degradation by white-rot fungi proceeds through oxidative depolymerization of lignin polymers, followed by metabolism of the resulting low-molecular-weight aromatic fragments. Among these, the catabolism of the hydroxyphenyl (H) unit of p-coumaric acid (p-CA) remains poorly understood in fungi. Here, we investigated the metabolism of p-CA by Trametes versicolor. Two group A flavoprotein monooxygenases (FPMOs), TvMNX3 and TvMNX4, catalyzed the hydroxylation of p-CA to caffeic acid. Recombinant expression and biochemical analyses revealed that TvMNX4 exhibited the highest catalytic efficiency toward both p-CA and 4-hydroxybenzoic acid, suggesting that it plays a major role in these hydroxylation reactions. Both enzymes also hydroxylated several p-CA derivatives, including 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol, as well as other lignin-derived guaiacyl and syringyl compounds. Structural modeling and docking analyses indicated that the substrate-binding pocket-particularly residue Leu219 in TvMNX4-is critical for substrate accommodation and catalytic activity. Together, these findings suggest that T. versicolor degrades p-CA via hydroxylation mediated by group A FPMOs. To our knowledge, this is the first report identifying p-CA 3-hydroxylase activity in eukaryotic FPMOs, expanding our understanding of fungal aromatic catabolism.

White-rot fungi are key players in the global carbon cycle through lignin degradation, yet the intracellular pathways that catabolize lignin-derived aromatics remain largely unresolved. The hydroxyphenyl unit compound p-coumaric acid (p-CA) is a major lignin fragment, but the enzymes responsible for its conversion to caffeic acid (CFA) have not been previously identified in fungi. This study demonstrates that Trametes versicolor employs group A flavoprotein monooxygenases (FPMOs) TvMNX3 and TvMNX4 for the hydroxylation of p-CA and related metabolites, representing an unrecognized branch of the p-CA catabolic pathway. Beyond ecological significance, the capacity of TvMNX4 to generate bioactive phenolics such as CFA and piceatannol underscores its potential for biotechnological applications, including the sustainable synthesis of pharmaceuticals and polymer precursors.