Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.
The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme.
D005292:Ferricyanides / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D013053:Spectrophotometry; D014414:Tungsten / Q000494:pharmacology; D019820:Xanthine; D014968:Xanthine Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D005292:Ferricyanides; D014970:Xanthines; C007931:hexacyanoferrate III; D019820:Xanthine; D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases; D014414:Tungsten