Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering

d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from <i>Thermoprotei</i> archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni²⁺. Its <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m values toward d-fructose were measured to be 248.5 min^-1 and 2.117 mM^-1·min^-1, respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a <i>t</i>_1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.