title: Purification and characterization of NADPH-dependent flavin reductase. An enzyme required for the activation of chorismate synthase in Bacillus subtilis.
abstract: NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D003509:Cyclohexanecarboxylic Acids; D004789:Enzyme Activation; D005486:Flavin Mononucleotide; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases; D014753:Vinyl Compounds
chemical_list: D003509:Cyclohexanecarboxylic Acids; D014753:Vinyl Compounds; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D008190:Lyases; C019895:chorismate synthase; D019761:Phosphorus-Oxygen Lyases