Expression, purification, and characterization of a membrane-bound D-amino acid dehydrogenase from Proteus mirabilis JN458.

OBJECTIVES: To characterize a novel membrane-bound D -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD).

RESULTS: The recombinant PmDAD protein, encoding a peptide of 434 amino acids with a MW of 47.7 kDa, exhibited broad substrate specificity with D -alanine the most preferred substrate. The K m and V max values for D -alanine were 9 mM and 20 μmol min-1  mg-1 , respectively. Optimal activity was at pH 8 and 45 °C. Additionally, this PmDAD generated H2 O2 and exhibited 68 and 60% similarity with E. coli K12 DAD and Pseudomonas aeruginosa DAD, respectively, with low degrees of sequence similarity with other bacterial DADs.

CONCLUSIONS: D-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.