Molekulare und kinetische Charakterisierung der Xanthin-Dehydrogenase aus dem phototrophen Bakterium Rhodopseudomonas capsulata / Molecular and Kinetic Characterization of Xanthine Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

Abstract

The structural and kinetic properties of xanthine dehydrogenase (EC 1.2.1.37) from the facultative phototrophic bacterium Rhodopseudomonas capsulata were studied. The enzyme was fully induced when hypoxanthine or xanthine, but less effectively when uric acid served as nitrogen source during growth. The enzyme was purified about 2300-fold from cells grown photosynthetically with hypoxanthine as N-source by using ammoniumsulfate precipitation, gel filtration, ion-exchange and affinity chromatography. The molecular weight as determined by gel filtration throug Sephacryl S-300 was 345000. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested a composition of four identical subunits with a molecular weight of 84000. The enzyme contained 2 flavin, 2 molybdenum and 8 iron-sulfur groups per mol. The turnover number with hypoxanthine and NAD as substrates was 12000 min^-1. Hypoxanthine, 1-methylhypoxanthine, 8-azahypoxanthine, xanthine, 1-methylxanthine, 2-hydroxypurine, 6,8-di-hydroxypurine, 5-azacytosine and 5-azauracil served as electron donors. The most effective electron acceptor was NAD. The kinetic constants (K_m) were (in μm): 52.5 (hypoxanthine); 32.5 (xanthine) and 61.2 (NAD). Various purine compounds inhibited the enzyme competitively in respect to hypoxanthine as substrate. Although reduction of uric acid to xanthine was not detected by using purified enzyme preparations, in vitro-experiments with ¹⁴C-labelled uric acid indicated that the enzyme xanthine dehydrogenase participates in uric acid degradation in Rps. capsulata. According to their electrophoretic mobilities, the xanthine dehydrogenases isolated from hypoxanthine-and uric acid-grown cells were identical.