The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1.
A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000032:analysis; D011549:Pseudomonas / Q000201:enzymology*; D011735:Pyridoxic Acid / Q000378:metabolism; D011736:Pyridoxine / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D005182:Flavin-Adenine Dinucleotide; D011092:Polyethylene Glycols; D011735:Pyridoxic Acid; D017830:Octoxynol; D000429:Alcohol Oxidoreductases; C038807:4-pyridoxic acid dehydrogenase; D011736:Pyridoxine