Improving the transallylation activity of halide methyltransferase from Arabidopsis thaliana through rational design

The allyl group is a ubiquitous structural motif found in numerous natural products and bioactive molecules. It can also serve as a highly reactive and versatile building block for the preparation of pharmaceuticals and functional materials in organic chemistry. Currently, the introduction of allyl groups mainly relies on chemical methods that require transition metals, especially precious ones. Although biocatalytic allylation using allylated S-adenosyl-L-methionine (SAM) as the allyl donor provides a promising, sustainable route to allylated compounds, the current access to allylated SAM is still not sufficiently efficient for further preparative-scale applications. In this study, we successfully engineered an Arabidopsis thaliana halide methyltransferase mutant named AtHMT A144G for the efficient synthesis of allylated SAM. Experimental results showed that the A144G variant (∼2.6 × 10^-5 s^-1 μM^-1) exhibited an ∼44-fold higher catalytic efficiency (k_cat/K_M) relative to the previously reported V140T mutant (∼5.9 × 10^-7 s^-1 μM^-1, newly measured in our study). In addition, the mutation did not alter the stereoselectivity of the enzyme, which still exclusively afforded the reactive (S)-diastereoisomer. To fully exploit the superior catalytic performance of A144G, we systematically optimized the reaction conditions, including the buffer system (KPi buffer), pH (7.5), and temperature (30 °C). With the optimal reaction conditions in hand, the feasibility of using AtHMT A144G as a catalyst to efficiently supply allylated SAM was validated in a cascade reaction with C/N/O-methyltransferases (e.g., NovO, COMT, and NMT) for allyl transfer. Furthermore, the desired allylated products were successfully obtained, confirming the practicality of A144G as an allyl donor platform for diverse downstream allyl transfer reactions.