The carboxylate “gripper” of the substrate is critical for C-4 stereo-inversion by UDP-glucuronic acid 4-epimerase

UDP-glucuronic acid 4-epimerase (UGAepi) catalyzes the NAD⁺-dependent interconversion of UDP-glucuronic acid (UDP-GlcA) and UDP-galacturonic acid (UDP-GalA) through a mechanism involving C4-oxidation, 4-keto-intermediate rotation, and subsequent reduction. Here, the functional significance of the substrate's carboxylate group in the epimerization process was investigated using UDP-4-keto-pentose, an analogous intermediate that lacks a carboxylate moiety. Site-directed mutations were introduced into UGAepi from Bacillus cereus (BcUGAepi) to increase substrate binding pocket flexibility, enabling the variant enzymes to accommodate UDP-4-keto-pentose more efficiently than the wild-type does. Although these BcUGAepi variants partially maintained nonstereospecific C4-epimerization activity with UDP-GlcA, they demonstrated fully stereospecific reduction of UDP-4-keto-pentose to UDP-xylose. These findings highlight the critical role of the carboxylate moiety as an essential element for epimerization in BcUGAepi, and elucidate the structural determinants of substrate specificity in UGAepis.