Functional characterization and catalytic mechanism of a C/N-selective methyltransferase MaMT4 involved in 1-deoxynojirimycin-type alkaloids biosynthesis in mulberry leaves

1-Deoxynojirimycin (DNJ)-type alkaloids represent the hallmark hypoglycemic bioactive components in mulberry leaves ( Morus alba L.). However, the methylation steps involved in their biosynthesis, particularly the roles and catalytic mechanism of plant C -methyltransferases, remain poorly understood. In this study, through further mining of transcriptome data, we screened and identified two methyltransferase genes significantly correlated with DNJ content in mulberry leaves ( P <0.05). Enzymatic assays demonstrated that MaMT4 catalyzes the methylation of piperidine at the C 2 position to form 2-methylpiperidine, while also mediating the N -methylation of piperidin-4-ol to generate 1-methylpiperidin-4-ol, revealing an unusual substrate-dependent C/N -position methylation selectivity. Enzyme kinetic analysis revealed that MaMT4 exhibits higher catalytic efficiency and substrate affinity for piperidine than toward piperidin-4-ol. Furthermore, in vivo gene overexpression and silencing experiments confirmed that MaMT4 positively regulates the accumulation of DNJ-type alkaloids in mulberry leaves. To elucidate the catalytic mechanism of MaMT4, molecular docking, site-directed mutagenesis, and in silico mutational analyses were performed. The results indicate that MaMT4 can modulate the relative contributions of hydrogen-bonding networks and hydrophobic interactions in response to subtle changes in substrate polarity and steric properties, thereby selectively recognizing and accommodating structurally diverse substrates. In particular, F363 and I80 were identified as critical residues responsible for this property. Together, these findings identify MaMT4 as a key methyltransferase involved in DNJ-type alkaloid biosynthesis and provide new insight into the catalytic mechanism and functional diversity of plant methyltransferases. • MaMT4 is a key methyltransferase involved in DNJ-type alkaloid biosynthesis. • MaMT4 shows rare substrate-dependent C/N methylation selectivity. • MaMT4 catalyzes C 2-methylation of piperidine and N -methylation of piperidin-4-ol. • In planta assays confirm the role of MaMT4 in DNJ-type alkaloid accumulation. • F363 and I80 are key residues affecting the catalytic activity of MaMT4.