Structural basis for oxidative decarboxylation of lignin-derived aromatics by the fungal flavoprotein monooxygenase PcMNX1.

Abstract

Lignin depolymerization by white-rot fungi generates diverse aromatic compounds derived from hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Although the metabolic pathways for G- and H-unit-derived aromatics have been studied, the enzymatic step responsible for the oxidative decarboxylation of the S-unit intermediate syringic acid (SA) has remained unknown. Here, we identify PcMNX1, a group A flavoprotein monooxygenase (FPMO) from the white-rot fungus Phanerochaete chrysosporium, as the enzyme catalyzing this missing step. Recombinant PcMNX1 catalyzed the NAD(P)H-dependent oxidative decarboxylation of SA to dimethoxyhydroquinone (DMHQ) and also converted other lignin-derived aromatics, including vanillic acid and 4-hydroxybenzoic acid, with markedly higher catalytic efficiency than the closely related enzyme GsMNX1 from Gelatoporia (Ceriporiopsis) subvermispora. The crystal structure of PcMNX1 was determined at 2.00 Å resolution, revealing a typical group A FPMO fold with FAD bound in the "out" conformation. Structure-guided mutagenesis demonstrated that His247 functions as the catalytic base required for decarboxylative hydroxylation. Comparative structural analysis with bacterial 3-hydroxybenzoate 6-hydroxylase (3HB6H) indicated that subtle substitutions in active-site residues alter substrate positioning and reaction outcomes. Consistent with this hypothesis, introduction of PcMNX1-type residues into 3HB6H conferred decarboxylation activity toward lignin-derived aromatics. Furthermore, enlargement of the PcMNX1 active-site cavity through the L264A substitution markedly enhanced SA conversion. Together, these findings demonstrate that PcMNX1 catalyzes the oxidative decarboxylation of SA and reveal how subtle active-site remodeling diversifies the catalytic repertoire of closely related group A FPMOs involved in lignin-derived aromatic metabolism.