D-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637.
Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. D-Amino acid dehydrogenase is a flavoenzyme that digests free neutral D-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 D-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial D-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q(1), thus distinguishing it from D-amino acid oxidase. The apparent K(m) and V(max) values were 40.2 mM and 25.0 micromol min(-1) mg(-1), respectively, for dehydrogenation of D-proline, and were 8.2 microM and 12.3 micromol min(-1) mg(-1), respectively, for reduction of Q(1). The respective pH and temperature optima were 8.0 and 37 degrees C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian D-amino acid oxidase than other bacterial D-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from D-proline to a c-type cytochrome was suggested spectrophotometrically.
Tanigawa
Minoru
M
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, Tokyo, 101-8308, Japan.
Shinohara
Tomomitsu
T
Saito
Makoto
M
Nishimura
Katsushi
K
Hasegawa
Yuichiro
Y
Wakabayashi
Sadao
S
Ishizuka
Morio
M
Nagata
Yoko
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D016480:Helicobacter pylori / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003605:D-Amino-Acid Oxidase