Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of D-mannose to D-glucose, whereas they did not act on β-(1→4)-mannobiose, N-acetyl-D-glucosamine, and D-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for D-mannose epimerization were 3.89 and 3.51 min −1 mM −1 , respectively. Monitoring the Runsl_4512 reaction through 1 H-NMR showed the formation of β-D-glucose and β-D-mannose from D-mannose and D-glucose, respectively. In the reaction with β-D-glucose, β-D-mannose was produced at the initial stage of the reaction, but not in the reaction with α-D-glucose. These results indicate that Runsl_4512 catalyzed the 2epimerization of the β-anomer substrate with a net retention of the anomeric configuration. Since 2 H was obviously detected at the 2-C position of D-mannose and D-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2 H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L D-glucose at 50°C and pH 8.0, Runsl_4512 and Dfer_5652 produced D-mannose with a 24.4 and 22.8% yield, respectively. These D-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing D-mannose.

Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of D-mannose and D-glucose, has recently been characterized to have potential for D-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME–D-glucitol and RsME(D254A)–D-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7–α8). The RsME–D-glucitol structure showed that loopα7–α8 moves towards D-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7–α8 are only conserved in MEs and interact with D-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)–D-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7–α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.