A stable NADP+-dependent D-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized D-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 µmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward D-leucine, D-norleucine, D-methionine, D-isoleucine, and D-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 µmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxoanalogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic D-amino acids with bulky side chains. In recent years, the potential uses for D-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of D-amino acids, but all include tedious steps. The use of NAD(P)+-dependent D-amino acid dehydrogenase (DAADH) makes single-step production of D-amino acids from oxoacid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of D-amino acids, such as D-leucine and D-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of D-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. [ABSTRACT FROM AUTHOR]