A halide methyltransferase family unites methyl-donor regeneration and methylation in a single scaffold
Abstract
The “magic methyl effect” is central to drug discovery, yet achieving precise methylation remains challenging. Methyltransferases offer high selectivity but are limited by inefficient methyl-donor regeneration. Halide methyltransferases (HMTs) in multienzyme cascades expand donor scope but suffer from complexity, cost, and poor inter-enzyme mass transfer. Here we report for the first time a halide methyltransferase from Aspergillus terreus (AteHMT) that directly couples methyl-donor regeneration with substrate methylation within a single enzyme using methyl iodide. Directed evolution enhances this noncanonical activity by ~100-fold. Structural analysis of donor–acceptor–enzyme complexes reveals that substrate-induced N-terminal loop reorganization enables productive acceptor binding and catalytic coupling. We further demonstrate that this bifunctionality is conserved across an HMT subfamily via sequence similarity network analysis and mutagenesis. Engineered variants enable efficient methylation of diverse substrates, establishing a general single-enzyme platform. This work efficiently collapses methyl-donor supply and transfer into one polypeptide, overcoming a long-standing limitation in biocatalytic methylation.
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