Oxygenation and Oxidation of Lignin Model Dimers by Fungal <i>Ortho</i> -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 <i>ortho</i>-methoxyphenolases (<i>o</i>-MPs, EC 1.14.18.13) and previously referred to as short polyphenol oxidases (PPOs), found in filamentous fungi can sequentially <i>o</i>-hydroxylate and oxidize guaiacyl-type phenols into methoxy-<i>o</i>-quinones. A subset of these enzymes also targets syringyl-type phenols and, via an unprecedented oxidative <i>o</i>-demethoxylation mechanism, funnels these into the same methoxy-<i>o</i>-quinones generated from guaiacyl-type compounds. Here, we demonstrate that fungal <i>o</i>-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-<i>o</i>-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 <i>o</i>-methoxyphenolases for catalyzing selective <i>ortho</i>-hydroxylation and two-electron oxidation of lignin components and provide a new foundation for developing enzyme-based lignin valorization strategies.