Proximal-to-distal molecular engineering of a halide methyltransferase for green synthesis of S-adenosyl-ethionine

Halide methyltransferases (HMTs) can efficiently catalyze the biosynthesis of S -adenosyl methionine (SAM) using natural substrates like methyl iodide or environmentally friendly alternatives such as methyl p-toluene sulfonate (MeOTs). Leveraging their broad substrate promiscuity, HMTs can also accommodate long-chain alkyl iodides and sulfonate esters, thereby facilitating the enzymatic production of structurally diverse SAM analogues. To address the limitations of existing S -adenosyl-ethionine (SAE) synthesis routes that depend on toxic alkyl halides like ethyl iodide, we developed a sustainable enzymatic strategy using ethyl p-toluene sulfonate (EtOTs) as the ethyl donor. Through a proximal-to-distal molecular engineering strategy, we systematically engineered wild-type Aspergillus clavatus halide methyltransferase ( Ac HMT) to enhance its catalytic properties. Following multiple rounds of semi-rational design, the mutant Ac HMT M4 exhibited a 6.0-fold increase in specific activity toward EtOTs compare to the wild-type, reaching 0.30 mU mg −1 , which represents the highest catalytic efficiency reported to date for this substrate. Molecular dynamics simulations subsequently revealed the structural mechanism of the improved catalytic activity. Biosynthesis of ethyl vanillin was achieved through a designed dual-enzyme cascade reaction. This work provides a universal strategy for engineering HMT to utilize sulfonate ester substrates, and an efficient biocatalyst for green synthesis of SAE for further ethyl modification of functional molecules.