Cobalamin‐Dependent O ‐Demethylation of Polymethoxyflavones by Enzymes Isolated From the Human Gut Microorganism Blautia producta MRG‐PMF1

O ‐demethylation of aryl methyl ethers is an invaluable transformation, playing a critical role in ecological circulation by degrading abundant lignin‐derived compounds and other natural products. Chemical O ‐demethylation requires harsh reaction conditions; thus, the need for biocatalytic solutions is of high interest. Here, we describe two cobalamin‐dependent enzyme systems from the human gut microorganism Blautia producta MRG‐PMF1, namely methyltransferase I (MT1) paired with corrinoid protein (CP), catalyzing the O ‐demethylation of polymethoxyflavones. The regioselectivity of the enzymes toward 10‐methoxyflavone molecules have been determined by HPLC–MS/MS analysis and NMR spectroscopy of chemically and enzymatically synthesized hemimethoxyflavones. One of the enzymes, BpMT1a, displayed processive O ‐demethylation in a regioselective manner, leading to complete demethylation of the substrates. Only certain hemimethoxylated intermediates accumulated during the reaction, indicating that certain intermediates remain bound in the active site during exchange of the CP. BpMT1b displayed strict regioselectivity toward O ‐demethylation of position 4′‐OMe of all the tested flavones. A photocatalytic regeneration system was implemented in scaled reactions with BpMT1b toward three methoxyflavones, allowing purification of a sufficient quantity of products for NMR characterization. With these findings, we have transitioned the use of cobalamin‐dependent O ‐demethylases from simple demethylation tools to a more sophisticated late‐stage functionalization platform for complex natural products.