Catalytic activity and substrate specificity of the engineered IEMT mutants. A, LC-MS profiling of the reactions catalyzed by IEMT (dashed line), and the mutant E165F (solid line) incubated with coniferyl alcohol and the methyl donor AdoMet. B, LC-MS profiling of the reactions catalyzed by IEMT (dashed line) and mutant E165F (solid line) incubated with sinapyl alcohol and the methyl donor AdoMet. C, relative activity of the selected IEMT mutant variants in the 4-O-methylation of coniferyl alcohol. 100% activity represents 147 ± 6 nmol mg−1 min−1. D, substrate specificity of the engineered monolignol 4-O-methyltransfeases (IEMT triple mutants). The error bars represent S.D. of three replicate.