Discovery of a\nThermostable Tagatose 4‑Epimerase\nPowered by Structure- and Sequence-Based Protein Clustering

d-Tagatose is a highly promising functional\nsweetener\nknown for its various physiological functions. In this study, a novel\ntagatose 4-epimerase from <i>Thermoprotei</i> archaeon (Thar-T4Ease),\nwith the ability to convert d-fructose to d-tagatose,\nwas discovered through a combination of structure similarity search\nand sequence-based protein clustering. The recombinant Thar-T4Ease\nexhibited optimal activity at pH 8.5 and 85 °C, in the presence\nof 1 mM Ni<sup>2+</sup>. Its <i>k</i><sub>cat</sub> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> values toward d-fructose were measured to be 248.5 min<sup>–1</sup> and 2.117 mM<sup>–1</sup>·min<sup>–1</sup>, respectively.\nNotably, Thar-T4Ease exhibited remarkable thermostability, with a <i>t</i><sub>1/2</sub> value of 198 h at 80 °C. Moreover,\nit achieved a conversion ratio of 18.9% using 100 g/L d-fructose\nas the substrate. Finally, based on sequence and structure analysis,\ncrucial residues for the catalytic activity of Thar-T4Ease were identified\nby molecular docking and site-directed mutagenesis. This research\nexpands the repertoire of enzymes with C4-epimerization activity and\nopens up new possibilities for the cost-effective production of d-tagatose from d-fructose.