Structural and Functional Insights into the Molecular Mechanism of Tagatose 4-Epimerase from Thermoprotei Archaeon

Tagatose 4-epimerase (T4Ease) has been demonstrated as a valuable enzyme for the bioproduction of d-tagatose, a low-calorie sugar substitute with various physiological benefits. The recently identified T4Ease from Thermoprotei archaeon (Thar-T4Ease) exhibits the highest naturally occurring C4-epimerization activity reported to date and is capable of catalyzing the interconversion of d-fructose and d-tagatose. In this study, we elucidated the high-resolution crystal structure of Thar-T4Ease and conducted a detailed investigation of its structural features. Structural analysis revealed that Thar-T4Ease adopts a conserved (β/α)₈ TIM-barrel fold as the core scaffold, similar to the tagaturonate-fructuronate epimerase, but features a unique α3' helix/loop region that covers the substrate-binding pocket. Key residues within the substrate-binding pocket were identified, including the putative catalytic residues Glu51 and Asp99, the metal-binding residues His100 and His318, the phosphorylation site Sep321, and other residues involved in substrate recognition. Their functional roles were confirmed through mutagenesis experiments. These findings provide crucial insights into the structure-function relationship of Thar-T4Ease, offering a foundation for future enzyme engineering efforts aimed at enhancing its performance for the cost-effective production of d-tagatose.