Specific Residues Expand the Substrate Scope and Enhance the Regioselectivity of a Plant O‐Methyltransferase

An isoeugenol 4‐O‐methyltransferase (IeOMT), isolated from the plant Clarkia breweri, can be engineered to a caffeic acid 3‐O‐methyltransferase (CaOMT) by replacing three consecutive residues. Here we further investigated functions of these residues by constructing the triple mutant T133M/A134N/T135Q as well as single mutants of each residue. Phenolics with different chain lengths and different functional groups were investigated. The variant T133M improves the enzymatic activities against all tested substrates by providing beneficial interactions to residues which directly interact with the substrate. Mutant A134N significantly enhanced the regioselectivity. It is meta‐selective or even specific against most of the tested substrates but para‐specific towards 3,4‐dihydroxybenzoic acid. The triple mutant T133M/A134N/T135Q benefits from these two mutations, which not only expand the substrate scope but also enhance the regioselectivity of IeOMT. On the basis of our work, regiospecific methylated phenolics can be produced in high purity by different IeOMT variants