D-Amino add dehydrogenase from Helicobacter pylori NCTC 11637

摘要

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 activityto D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q1, thus distinguishing it from D-amino acid oxidase. The apparent K_m and V_(max) values were 40.2 mM and 25.0 umol min~(-1) mg~(-1), respectively, for dehydrogenation of D-proline, and were 8.2 μM and 12.3 umol min~(-1) mg~(-1) respectively, for reduction of Q1. The respective pH and temperature optima were 8.0 and 37°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.