Ancestral Protein Reconstruction Uncovers a Thermotolerant Rieske Oxygenase with Enhanced O -Demethylation Activity toward 3- O -Methylgallate

Biological lignin valorization offers a renewable route to value-added chemicals, yet its industrial implementation is bottlenecked by the inherent instability and low efficiency of O-demethylating enzymes. Here, we employ ancestral sequence reconstruction (ASR) to resurrect highly stable and efficient variants of VanA-type Rieske nonheme iron oxygenases, which are notoriously difficult to engineer. Our lead ancestral variant, AncVanA3, exhibits an unprecedented ∼30 °C increase in melting temperature (67 °C) and a 7-fold improvement in soluble yield compared to the extant XacVanA. Crucially, AncVanA3 achieves superior catalytic conversion of the recalcitrant lignin monomer 3-O-methylgallate (3OMG), reaching 92% conversion in the syringate-to-gallate funneling pathway. Molecular dynamics simulations reveal that this enhanced activity stems from the disruption of a gating mechanism involving a V300 gate-keeper residue, resulting in a constitutively open and enlarged substrate-binding pocket. Most significantly, biophysical characterization via mass photometry reveals that these enzymes exist predominantly as monomers in solution. This finding challenges the prevailing view that Rieske oxygenases self-assemble into intrinsically stable homotrimers to facilitate intersubunit electron transfer. Collectively, these results provide both a robust technological platform for lignin upgrading and a new mechanistic framework for the study of electron transfer in bioinorganic catalysis.