title: Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.
abstract: A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created fromUreibacillus 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.IMPORTANCEIn 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.
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Hayashi
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Seto
authors.affiliation: Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Hironaga
authors.initials: H
authors.lastname: Akita
authors.affiliation: Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Higashi-Hiroshima, Hiroshima, Japan.
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Hoshino
authors.affiliation: Department of Bioscience, School of Agriculture, Tokai University, Higashi-ku, Kumamoto-shi, Kumamoto, Japan.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Yoneda
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Asahi-ku, Osaka, Japan.
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.affiliation: Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kita-gun, Kagawa, Japan sakuraba@ag.kagawa-u.ac.jp.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
mesh_terms: D020816:Amino Acid Motifs; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D058641:Planococcaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009249:NADP; D000594:Amino Acid Oxidoreductases
keywords: NADP; Ureibacillus thermosphaericus; d-amino acid; d-phenylalanine; dehydrogenases; meso-diaminopimelate; phenylpyruvate