Selenium-containing xanthine dehydrogenase from Eubacterium barkeri

A specific dehydrogenase, different from nicotinic acid hydroxylase, was in duced during growth of Eubacterium barkeri on xanthine. The protein designa ted as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent spec ific activity of 164 mu mol 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/P AGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identif ied as the molybdenum-complexing cofactor using activity reconstitution exp eriments and fluorescence measurements after KI/I-2 oxidation. The molecula r mass of the cofactor indicated that it is of the dinucleotide type. The e nzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium an d FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of nati ve enzyme. Xanthine dehydrogenase was inactivated upon incubation with arse nite, cyanide and different purine analogs. Reconstitution experiments of x anthine dehydrogenase activity by addition of selenide and selenite perform ed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a c ovalently bound form such as selenocysteine.