Engineering of an Fe2+/α-Ketoglutarate-Dependent Dioxygenase Enables Stereoselective and Efficient Sulfoxide Synthesis

Alliin (S-allyl-l-cysteine sulfoxide) is a valuable bioactive sulfur compound and an allicin precursor. However, its biocatalytic synthesis is limited by poor stereocontrol of an Fe2+/α-ketoglutarate-dependent dioxygenase (l-isoleucine dioxygenase, IDO) that converts S-allyl-l-cysteine (SAC) into alliin. Herein, we analyzed the catalytic mechanism of IDO and proposed that the dihedral orientation of the SAC sulfur atom relative to the Fe(IV)═O intermediate likely governs sulfoxide chirality. Based on this mechanistic insight, we identified residue T233 and the flexible N77–F98 entrance loop as key determinants of substrate orientation during sulfoxidation. The engineered variant IDOM5 exhibited substantially enhanced diastereomeric excess (de, 93.4%) and turnover compared with the wild-type enzyme (de, 80.2%). Molecular dynamics simulations confirmed that the two geometric parameters encode stereochemical outcome and reaction rate, respectively, validating our initial hypothesis. Gram-scale biocatalytic synthesis of highly diastereomerically enriched (+)-alliin was achieved (2.70 g·L–1·h–1, conversion 93.70%). This work establishes a robust enzymatic platform for stereoselective alliin synthesis.