title: Creation of a thermostable NADP⁺-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis.
abstract: A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.
authors.affiliation: Applied Molecular Microbiology and Biomass Chemistry, Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. h.akita.117@s.kyushu-u.ac.jp
authors.forename: Hironaga
authors.initials: H
authors.lastname: Akita
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawarabayasi
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003641:Deamination; D004926:Escherichia coli; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D058641:Planococcaceae / Q000201:enzymology*; D017354:Point Mutation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003067:Coenzymes; D011994:Recombinant Proteins; D009249:NADP; D000594:Amino Acid Oxidoreductases; C020032:diaminopimelate dehydrogenase; D003605:D-Amino-Acid Oxidase