Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericusmeso-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 oxo-analogue) 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.IMPORTANCE 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 oxo-acid 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.

Structural insight into the thermostable NADP(+)-dependent meso-diaminopimelate dehydrogenase from Ureibacillus thermosphaericus.

Crystal structures of the thermostable meso-diaminopimelate dehydrogenase (DAPDH) from Ureibacillus thermosphaericus were determined for the enzyme in the apo form and in complex with NADP(+) and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. The main-chain coordinates of the enzyme showed notable similarity to those of Symbiobacterium thermophilum DAPDH. However, the subunit arrangement of U. thermosphaericus DAPDH (a dimer) was totally different from that of the S. thermophilum enzyme (a hexamer). Structural comparison with the dimeric enzyme from the mesophile Corynebacterium glutamicum revealed that the presence of large numbers of intrasubunit and intersubunit hydrophobic interactions, as well as the extensive formation of intersubunit ion-pair networks, were likely to be the main factors contributing to the higher thermostability of U. thermosphaericus DAPDH. This differs from S. thermophilum DAPDH, within which the unique hexameric assembly is likely to be responsible for its high thermostability. Analysis of the active site of U. thermosphaericus DAPDH revealed the key factors responsible for the marked difference in substrate specificity between DAPDH and the D-amino acid dehydrogenase recently created from DAPDH by introducing five point mutations [Akita et al. (2012). Biotechnol. Lett. 34, 1693-1699; 1701-1702].