Functional characterization and protein engineering of a O-methyltransferase involved in benzylisoquinoline alkaloid biosynthesis of Stephania tetrandra

Benzylisoquinoline alkaloids (BIAs) are the primary active components of Stephania tetrandra. However, the molecular mechanisms underlying BIA biosynthesis in S. tetrandra remain poorly understood. Here, we reported the isolation and characterization of a novel O-methyltransferase, designated as St7OMT1, from S. tetrandra. St7OMT1 exhibited strict substrate specificity and regioselectivity, catalyzing the conversion of (S)-coclaurine to norarmepavine at the C7 position. Optimal enzymatic activity of St7OMT1 was detected at 30 degrees C and pH 6.0 in sodium citrate buffer, with kinetic parameters of Km = 163.16 mu M and kcat = 0.05*10-3 s-1. Site-directed mutagenesis identified three critical residues Ala118, Gln121 and Arg151 that significantly influenced catalytic activity and substrate selectivity. A triple mutant (A118V-Q121L-R151L) was engineered, exhibiting a 5.97-fold increase in catalytic activity compared to the wild-type enzyme. Molecular dynamics simulations revealed that the enhanced catalytic activity resulted from an expanded substrate access tunnel and binding pocket, as well as improved non-covalent interactions between the enzyme and substrate. This study provides a promising St7OMT1 variant for the BIA biosynthesis with reduced by-product formation and offers valuable insights into protein engineering strategies to enhance O-methyltransferase activity in plants.