Selenium-containing xanthine dehydrogenase from Eubacterium barkeri.
A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent specific activity of 164 micromol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH-dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum-complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.
Schräder
T
T
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Germany.
Rienhöfer
A
A
Andreesen
J R
JR
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D003429:Cross Reactions; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005051:Eubacterium / Q000201:enzymology* / Q000254:growth & development; D007120:Immunochemistry; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D012643:Selenium / Q000737:chemistry* / Q000378:metabolism; D018038:Sodium Selenite / Q000494:pharmacology; D013053:Spectrophotometry; D013379:Substrate Specificity; D017973:Tungsten Compounds / Q000494:pharmacology; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000907:Antibodies, Bacterial; D004791:Enzyme Inhibitors; D017973:Tungsten Compounds; C044659:molybdate; D009249:NADP; D008982:Molybdenum; D014968:Xanthine Dehydrogenase; D012643:Selenium; D018038:Sodium Selenite; C045951:tungstate