Regioselectivity Inversion of an O‐Methyltransferase via Semi‐rational Mutagenesis Combined with Metal Ion Substitution

Abstract S ‐adenosyl‐L‐methionine (SAM)‐dependent O ‐methyltransferases ( O ‐MTs) are a class of valuable and attractive biocatalysts for alkylation of catechols. In this work, an O ‐methyltransferase ( Ge OMT) from the plant Galanthus elwesii was identified and characterised. Ge OMT methylated a series of catechols with excellent regioselectivity. Moreover, a strategy was developed that combines structure‐directed protein engineering and metal ion substitution for regioselective inversion of Ge OMT. The resultant W50M/A53N/Y186K triple variant preferentially coordinated Ni 2+ instead of Mg 2+ and displayed almost complete inversion in regioselectivity from 1 : 99 to 94 : 6 ( para / meta ) for 4‐nitrocatechol, the para ‐methylated product of which is a vital plant growth regulator. This represents a promising strategy for regioselectivity improvement of O ‐MTs.