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

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.