Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases. which catalyze the last two steps in th e formation of urate, are synthesized as the dehydrogenase form xanthine de hydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here. we present the crystal structure of the dimeric (M-r, 290.000) bovine milk XDH at 2.1-Angstrom resolution and XO at 2.5-Angstrom resolution and descri be the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain cont aining two iron sulfur centers, a central 40-kDa flavin adenine dinucleotid e domain. and a C-terminal 85-kDa molybdopterin-binding domain with the fou r redox centers aligned in an almost linear fashion. Cleavage of surface-ex posed loops of XDH causes major structural rearrangement of another loop cl ose to the flavin ring (Gln 423-Lys 433). This movement partially blocks ac cess of the NAD substrate to the flavin adenine dinucleotide cofactor and c hanges the electrostatic environment of the active site, reflecting the swi tch of substrate specificity observed for the two forms of this enzyme.