Tailoring the Regioselectivity of <i>Lentinula edodes</i> <i>O</i>-Methyltransferases for Precise <i>O</i>-Methylation of Flavonoids
Jean‐Philippe Kanter, Meike Ahlhorn, Holger Zorn, Binglin Li, Martin Gand
Abstract
High Resolution Image Download MS PowerPoint Slide A novel O -methyltransferase, Le OMT4, from Lentinula edodes was identified, expressed, and characterized. Although its catalytic activity was lower than that of the previously reported Le OMT2, Le OMT4 displayed strong regioselectivity for the meta -hydroxy group across different catecholic compounds, producing e.g., ferulic acid with an almost exclusive regioisomeric ratio of 98:2 and homoeriodictyol with a ratio of 82:18 (3′-product:4′-product). Leveraging the high sequence and predicted structural similarity between Le OMT2 and Le OMT4, key sites for the tailoring of Le OMT2 were identified through site-directed mutagenesis. This approach aimed for robust mutants retaining the high specific activity of Le OMT2, while enhancing regioselectivity. A single amino acid substitution, F182Y, enabled a regioisomeric ratio of 91:9 for the production of homoeriodictyol. Notably, another single amino acid substitution, I53M reversed the regioselectivity to 2:98 in favor of hesperetin. This strategy enables the selective production of sought-after pharmacologically active flavonoids (butein) and flavor-active flavonoids (homoeriodictyol, hesperetin, hesperetin dihydrochalcone).