Purification and properties of D-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur flavoenzyme from Escherichia coli B.

D-Amino acid dehydrogenase, a membrane-associated oxidative enzyme which couples D-alanine oxidation to solute active transport in Escherichia coli B cytoplasmic membrane vesicles, has been solubilized with 0.1% (W/V) Triton X-100 and purified 120-fold from these membranes in the presence of 0.02% Triton X-100 to 265% homogeneity.As isolated, there are 0.8 mg of Triton X-100/mg of enzyme protein.Analytical isoelectric focusing gels show one major band with enzymic activity; a second dimension electrophoresis in the presence of sodium dodecyl sulfate indicates two subunits with molecular weights of approximately 55,000 and 45,000.The enzyme contains flavin adenine dinucleotide and non-heme iron in a ratio of 1:2 to 3, consistent with the absorption spectrum of this yellow enzyme in the 400-to 500-nm region.The purified enzyme will oxidize several D-amino acids with kinetic constants similar to that of the membrane bound enzyme, but it cannot use molecular oxygen as an electron acceptor.Instead, dichlorophenolindophenol and coenzyme Q analogues can serve as electron acceptors.In addition, coenzyme Q 1 can mediate electron transfer to other acceptors.With dichlorophenolinophenol, coenzyme Q1 stimulates V,,, rates for dye reduction %fold; with a coenzyme Q1-potassium ferricyanide couple, a V,, of 38.0 units/mg is estimated for homogeneous enzyme, corresponding to a turnover number of approximately 4,400/min/enzyme-bound flavin adenine dinucleotide.Oxidation of ~-[2-'HJalanine yields a V,,, isotope effect of approximately 1.9, similar to that seen in cytoplasmic membrane vesicles, suggesting no change in rate-determining step upon solubilization and purification.D-Amino acid dehydrogenase is a bacterial membrane enzyme that catalyzes the oxidation of a variety of D-amino acids