d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Abstract

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.

Purification of Helicobacter pylori NCTC 11637 Cytochrome bc 1 and Respiration with d -Proline as a Substrate

“…The moderate activity with these amino acids may be ascribed to the large K m value of the DAD-mediated reaction with the best substrate, D-proline (17), although the K m value is comparable to that of E. coli enzyme (14). The reaction was carried out at pH 7.0, while the optimum pH of DAD is 8.0 (17) because almost no oxygen uptake was observed at pH 8.0, which may be due to other relevant reactions repressed at pH 8.0.…”

“…DAD was purified from recombinant E. coli cells harboring the H. pylori NCTC 11637 dadA gene (accession number AB295062) according to a previously reported method (17).…”

“…Since the intrinsic quinone of H. pylori, menaquinone-6 was not commercially available, we used Q 0 . Upon the addition of D-proline, the most active substrate of DAD (17), to the reconstituted respiratory system, a reduced type of absorption spectrum of cytochrome cbb 3 appeared (Fig. 4A), although the height of the ␣ peak was lower than that reduced with hydrosulfite (Fig.…”

“…DAD has been known to occur in Gram-negative Gammaproteobacteria, such as facultatively anaerobic Escherichia coli (15) and Salmonella typhimurium (20), and bacteria such as Pseudomonas fluorescens (18) and P. aeruginosa (11) that are able to grow using nitrate as a final electron acceptor in anaerobic respiration. We detected DAD activity in an obligate anaerobe, Pyrobaculum islandicum (13), and purified H. pylori DAD from the recombinant E. coli cells (17). The enzyme has not been reported in obligatory aerobic organisms such as vertebrates and higher plants.…”

Methemoglobin reduction mediated by d-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae

“…DAD has been found in organisms that inhabit oxygen-deficient environments, whereas D-amino acid oxidase, another FAD-containing enzyme with functions similar to DAD, is found in aerobic organisms. DAD has only been reported in bacteria such as Escherichia coli (Franklin and Venables, 1976), Pseudomonas fluorescens (Tsukada, 1966), Pseudomonas aeruginosa (Marshall and Sokatch, 1968), Salmonella typhimurium (Wild et al, 1974), and Helicobacter pylori (Tanigawa et al, 2010). These bacteria can inhabit microaerobic environments.…”