To characterize a novel membrane-bound d -amino acid dehydrogenase from Proteus mirabilis JN458 (PmDAD). 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 mu mol min(-1) mg(-1), respectively. Optimal activity was at pH 8 and 45 A degrees C. Additionally, this PmDAD generated H2O2 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. d-Amino acid dehydrogenase from Proteus mirabilis JN458 was expressed and characterized for the first time, DAD was confirmed to be an alanine dehydrogenase.