Substrate orientation in xanthine oxidase: crystal structure of enzyme in reaction with 2-hydroxy-6-methylpurine.
Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3A. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the MoV transition state.
Pauff
James M
JM
Department of Biochemistry, University of California, Riverside, California 92521, USA.
Zhang
Jinjin
J
Bell
Charles E
CE
Hille
Russ
R
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D006801:Humans; D033461:Hyperuricemia / Q000201:enzymology*; D008982:Molybdenum / Q000737:chemistry* / Q000378:metabolism; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011687:Purines / Q000737:chemistry* / Q000378:metabolism; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism
D011687:Purines; D009243:NAD; C071987:2-hydroxy-6-methylpurine; D008982:Molybdenum; D014969:Xanthine Oxidase