Oxygenation and Oxidation of Lignin Model Dimers by Fungal Ortho-Methoxyphenolases

Lignin is the largest
renewable resource for aromatics, and the
quest to understand enzymatic lignin modification has never been more
important. A recently recognized group of single-domain type-3 copper
enzymes, named ortho-methoxyphenolases (o-MPs, EC 1.14.18.13) and previously referred to as short polyphenol
oxidases (PPOs), found in filamentous fungi can sequentially o-hydroxylate and oxidize guaiacyl-type phenols into methoxy-o-quinones. A subset of these enzymes also targets syringyl-type
phenols and, via an unprecedented oxidative o-demethoxylation
mechanism, funnels these into the same methoxy-o-quinones
generated from guaiacyl-type compounds. Here, we demonstrate that
fungal o-methoxyphenolases also cleave bonds in lignin
model dimers representing the abundant β-O-4′-linked
substructures of lignin, having guaiacyl and, in some cases, syringyl
terminal phenolic groups. Based on advanced liquid chromatography-mass
spectrometry (LC-MS), nuclear magnetic resonance (NMR) analysis, and
isotope labeling, we propose a mechanism in which the enzymatic formation
of methoxy-o-quinone moieties in the model dimers
triggers intramolecular rearrangements that lead to different types
of bond cleavage, where C1–Cα cleavage predominates.
Additionally, β-ether breakage and formation of Cα-ketone
groups occur. We investigate the influence of pH and reductants on
reaction pathways and identify strategies to steer the reaction toward
either depolymerization or oxyfunctionalization of the dimers without
interunit bond cleavage. The enzymes also target Cα-oxidized
model dimers, albeit at lower rates. The findings of this study demonstrate
the potential of using fungal o-methoxyphenolases
for catalyzing selective ortho-hydroxylation and
two-electron oxidation of lignin components and provide a new foundation
for developing enzyme-based lignin valorization strategies.