Novel Methylenetransferase from Human Gut Strain <i>L. longoviformis</i> Initiates Sesamin Conversion and Its Catalytic Mechanism

Gut microbiota governs lignan bioactivation, yet the specific enzymes involved remain poorly characterized. Here, we identified a novel sesamin methylenetransferase from <i>Lactonifactor longoviformis</i>, <i>Lac</i>AMT, that catalyzes the methylenedioxy bridge cleavage of sesamin (SESA) into catechol metabolites, with <i>K</i>_m and <i>k</i>_cat values of 1.203 mM and 0.83 min^-1. <i>Lac</i>AMT homologues are rare in human gut microbes, mainly in the Lachnospiraceae and Clostridiaceae families. Structural and mutational analyses revealed that <i>Lac</i>AMT employs a unique catalytic mechanism distinct from those of typical GcvT methyltransferases and hepatic P450s. <i>Lac</i>AMT cleaves SESA's methylenedioxy bridge and transfers the methylene group to tetrahydrofolate through Y218 and H222, acting as an acid-base pair. Moreover, enhanced substrate binding with Y218 likely facilitates proton transfer, resulting in 3.33-fold and 2.12-fold increases in catalytic efficiency for W107E and V94A, respectively. These findings elucidate how the human gut microbiome activates plant secondary metabolites, advancing our understanding of the interplay among the diet, gut microbiota, and human health.