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

• Published in: Applied and environmental microbiology 2017-06, Vol.83 (11)
• Main Authors: Hayashi, Junji, Seto, Tomonari, Akita, Hironaga, Watanabe, Masahiro, Hoshino, Tamotsu, Yoneda, Kazunari, Ohshima, Toshihisa, Sakuraba, Haruhiko
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Oxidoreductases - chemistry; Amino Acid Oxidoreductases - genetics; Amino Acid Oxidoreductases - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Enzymology and Protein Engineering; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; NADP - metabolism; Planococcaceae - chemistry; Planococcaceae - enzymology; Planococcaceae - genetics; Protein Engineering; Substrate Specificity
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.00491-17

A stable NADP -dependent d-amino acid dehydrogenase (DAADH) was recently created from -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. 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