Enterococcus faecium NAD-dependent D-mandelate dehydrogenase (D-ManDH) belongs to a ketopantoate reductase (KPR)-related D-2-hydroxyacid dehydrogenase family, and exhibits broad substrate specificity toward bulky hydrophobic 2-ketoacids, preferring C3-branched substrates. The ternary complex structure of D-ManDH with NADH and anilino( oxo)acetate (AOA) revealed that the substrate binding induces a shear motion of the N-terminal domain along the C-terminal domain, following the hinge motion induced by the NADH binding, and allows the bound NADH molecule to form favorable interactions with a 2-ketoacid substrate. D-ManDH possesses a sufficiently wide pocket that accommodates the 0 branched side chains of substrates like KPR, but unlike the pocket of KPR, the pocket of D-ManDH comprises an entirely hydrophobic surface and an expanded space, in which the AOA benzene is accommodated. The expanded space mostly comprises a mobile loop structure, which likely modulates the shape and size of the space depending on the substrate. (C) 2017 Elsevier Inc. All rights reserved.