Structural insights into the enzyme mechanism of a new family of d-2-hydroxyacid dehydrogenases, a close homolog of 2-ketopantoate reductase.

A newly identified family of NAD-dependent D-2-hydroxyacid dehydrogenases (D-2-HydDHs) catalyzes the stereo-specific reduction of branched-chain 2-keto acids with bulky hydrophobic side chains to 2-hydroxyacids. They are promising targets for industrial/practical applications, particularly in the stereo-specific synthesis of C3-branched D-hydroxyacids. Comparative modeling and docking studies have been performed to build models of the enzyme-cofactor-substrate complexes and identify key residues for cofactor and substrate recognition. To explore large conformational transitions (domain motions), a normal mode analysis was employed using a simple potential and the protein models. Our analysis suggests that the new D-2-HydDH family members possess the N-terminal NAD(H) binding Rossmann-fold domain and the alpha-helical C-terminal substrate binding domain. A hinge bending motion between the N- and C-terminal domains was predicted, which would trigger the switch of the conserved essential Lys to form a key hydrogen bond with the C2 ketone of the 2-keto acid substrates. Our findings will be useful for site-directed mutagenesis studies and protein engineering.