Human Xanthine Oxidase Changes its Substrate Specificity to Aldehyde Oxidase Type upon Mutation of Amino Acid Residues in the Active Site: Roles of Active Site Residues in Binding and Activation of Purine Substrate

Yamaguchi, Yoshihiro; Matsumura, Tomohiro; Ichida, Kimiyoshi; Okamoto, Ken; Nishino, Tokuzo

doi:10.1093/jb/mvm053

Cited by 126 publications

(164 citation statements)

References 48 publications

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“…The crystals examined in the present study were monoclinic in space group P2 1 , unlike previous structures of XOR but similar to recent work by Yamaguchi and coworkers (26). The overall dimensions of the unit cell were a ϭ 133.2 Å, b ϭ 73.8 Å, and c ϭ 146.5 Å with angles of 90, 98.9, and 90° (Table 1).…”

Section: Overall Structure Of Xo With 2-hydroxy-6-methylpurine-supporting

confidence: 82%

Substrate Orientation in Xanthine Oxidase

Pauff

Zhang

Bell

et al. 2008

Journal of Biological Chemistry

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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 O 2 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 2.3 Å . 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 Mo V transition state.Xanthine oxidoreductase (XOR) 2 is the prototypical member of the molybdenum hydroxylase family of proteins (1, 2). In humans, XOR catalyzes the hydroxylation of hypoxanthine to xanthine as well as xanthine to uric acid, and the mammalian enzyme exists in two alternative forms of the same gene product. Normally, the enzyme exists in a dehydrogenase form (xanthine dehydrogenase) but can be readily converted to an oxidase form (XO) by oxidation of sulfhydryl residues or by limited proteolysis (3). Xanthine dehydrogenase shows a preference for NAD ϩ as the oxidizing substrate (although it is also able to react with O 2 ), whereas XO is unable to react with NAD ϩ and uses O 2 exclusively (3). This conversion of xanthine dehydrogenase to XO is thought to be relevant in the context of ischemia-reperfusion pathology (4). The enzyme is a target of drugs against gout and hyperuricemia and is often targeted in tandem in chemotherapeutic regimens (5). Excellent reviews describing XOR in pharmacology and human pathology are available (6, 7).Like the human enzyme, the bovine enzyme is a 290-kDa homodimer, each monomer having a molybdenum center plus two [2Fe-2S] clusters (with each iron coordinated by a pair of cysteines) and FAD. Oxidative hydroxylation of the purine substrate occurs at the molybdenum center, which results in the two-electron reduction of the enzyme. After internal electron transfer via the [2Fe-2S] centers to the FAD, reducing equivalents are passed to the final electron acceptor (O 2 or NAD ϩ ). The crystal structure of the bovine enzyme has been determined (8), and it shows that the four redox-active centers of each monomer are found in separate, distinctly folding domains.The catalytic sequence of XOR is thought...

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Section: Overall Structure Of Xo With 2-hydroxy-6-methylpurine-supporting

confidence: 82%

Substrate Orientation in Xanthine Oxidase

Pauff

Zhang

Bell

et al. 2008

Journal of Biological Chemistry

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“…Mutation of Arg 310 to Met also results in a large reduction in k red , by a factor of 10 4 , an effect attributed to Arg 310/880 stabilizing negative charge on the heterocycle in the course of nucleophilic attack (11). The E232A variant exhibited a more modest 12-fold decrease in k red and a 12-fold increase in K d in the reductive half-reaction, relative to wild-type enzyme, comparable with the effect seen on the steady-state kinetic behavior of an E803V variant of the human enzyme (13). Given the effect of the mutation on both K d and k red , it is evident that Glu 232 is involved with both substrate binding and transition state stabilization, although its specific role has been somewhat controversial.…”

mentioning

confidence: 74%

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

Hall

Reschke

Cao

et al. 2014

Journal of Biological Chemistry

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“…Addendum-Since our manuscript was submitted, Nishino and coworkers (16) have reported the properties of several mutants of human xanthine oxidoreductase, including an R881M mutant analogous to the R310M mutant considered here with the R. capsulatus xanthine dehydrogenase. By steady-state kinetic analysis, these authors find no detectable activity in the mutant, consistent with our observation here that k red is reduced by a factor of 20,000.…”

Section: Acknowledgment-we Thank Antje Schulte For Construction Of Thmentioning

confidence: 99%

The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus

Pauff

Hemann

Jünemann

et al. 2007

Journal of Biological Chemistry

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The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k red (the limiting rate of enzyme reduction by substrate at high [S]) decreased ϳ20-fold in an R310K variant and 2 ؋ 10 4 -fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed ϳ4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k red greater than 10 3 ), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k red less than 50-fold). The effect was such that the 4000-fold range in k red seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.The molybdenum-containing hydroxylases represent a unique solution to the hydroxylation of carbon centers. Other monooxygenases introduce an oxygen atom derived from O 2 and consume two reducing equivalents (along with the two removed from the substrate to be hydroxylated) in reducing O 2 to water. The molybdenum enzymes, on the other hand, utilize water itself as the ultimate source of the oxygen atom incorporated into product. In the case of enzymes such as xanthine dehydrogenase, not only are molybdenum enzyme reducing equivalents not consumed in carrying out the catalyzed reaction, but in fact physiologically useful reducing equivalents in the form of NADH are generated. As such, these enzymes represent a unique solution to the chemistry of hydroxylation, and the requisite cleavage of a carbon-hydrogen bond that accompanies it (1).The xanthine dehydrogenase from Rhodobacter capsulatus is an (␣␤) 2 heterotetramer comprising two cop...

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Human Xanthine Oxidase Changes its Substrate Specificity to Aldehyde Oxidase Type upon Mutation of Amino Acid Residues in the Active Site: Roles of Active Site Residues in Binding and Activation of Purine Substrate

Cited by 126 publications

References 48 publications

Substrate Orientation in Xanthine Oxidase

Substrate Orientation in Xanthine Oxidase

The Reductive Half-reaction of Xanthine Dehydrogenase from Rhodobacter capsulatus

The Role of Arginine 310 in Catalysis and Substrate Specificity in Xanthine Dehydrogenase from Rhodobacter capsulatus

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