Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp 2 -hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 Å resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 Å resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg 880 and Glu 802 , previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.Purine oxidation in nature is catalyzed by three distinct classes of enzymes: the molybdenum-containing hydroxylases such as xanthine oxidoreductase (1, 2), the Fe II -and ␣-ketoglutarate-dependent xanthine hydroxylases (3, 4), and a newly described two-component system, HpxDE, consisting of a [2Fe-2S]/flavin-containing reductase and a Rieske/non-heme iron-containing oxygenase (5, 6). The first class is by far the most broadly distributed, with members found throughout the eubacteria, archaea, and eukaryota. The second class is found principally in fungae, including yeasts (Saccharomyces cerevisiae, for example), and the third is identified to date only in Klebsiella oxytoca and Klebsiella pneumoniae.Xanthine oxidoreductases from eukaryotes are homodimers of ϳ290 kDa, with each monomer containing four redox-active sites: an active site molybdenum center, a pair of spinach ferredoxin-like [2Fe-2S] clusters, and FAD (7). The overall catalytic sequence consists of a reductive half-reaction in which substrate is oxidatively hydroxylated at the molybdenum center (reducing it from Mo(VI) to Mo(IV)) and, after intramolecular electron transfer, an oxidative half-reaction in which reducing equivalents are removed from the enzyme via its FAD. In the reductive half-reaction, purine substrates are hydroxylated at a specific carbon position in a reaction initiated by nucleophilic attack of an equatorial Mo-OH group of the metal center whose deprotonation is thought to be facilitate...

Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in the course of reaction with the pterin substrate lumazine at 2.2 Å resolution and of the nonfunctional desulfo form of the enzyme in complex with xanthine at 2.6 Å resolution. In both cases the orientation of substrate is such that the pyrimidine subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.

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...

Xanthine oxidoreductase (XOR) is a molybdenum-containing enzyme that under physiological conditions catalyzes the final two steps in purine catabolism, ultimately generating uric acid for excretion. Here we have investigated four naturally-occurring compounds that have been reported to be inhibitors of XOR in order to examine the nature of their inhibition utilizing in vitro steadystate kinetic studies. We find that luteolin, silibinin, and quercetin are mixed-type inhibitors of the enzyme in vitro and, unlike allopurinol, the inhibition is not time-dependent. These three natural products also decrease the production of superoxide by the enzyme. In contrast, and contrary to previous reports in the literature based on in vivo and other non-mechanistic studies, we find that curcumin did not inhibit the activity of purified XO, nor its superoxide production in vitro.Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-containing enzyme that has been studied extensively from a biochemical perspective for more than 80 years. In human physiology, XOR catalyzes the final two steps of purine catabolism, transforming hypoxanthine to xanthine and then xanthine to uric acid by sequential oxidative hydroxylations at C-2 and C-8 of the purine ring, respectively ( Figure 1). 1 Xanthine oxidoreductase exists in two forms. The protein normally exists as a dehydrogenase (xanthine dehydrogenase, XDH), and utilizes NAD + as its final electron acceptor in catalysis. Under certain conditions, most notably ischemia and/or hypoxia, XDH can be converted to an oxidase form (XO), which can no longer reduce NAD + and instead utilizes O 2 exclusively as the terminal electron acceptor in the course of turnover. This conversion may occur either by oxidation of sulfhydryl groups and/or by limited proteolysis. 2, 3 Once in the oxidase form, the enzyme generates significant amounts of H 2 O 2 and superoxide radicals, although XDH can also react with O 2 and generate these reactive oxygen species (ROS). 3 Under normal conditions, however, NAD + effectively competes with O 2 and limits the generation of ROS by the dehydrogenase. 6,7 The specific conformational change responsible for the D to O conversion involves modification of the access channel to the flavin site (with its FAD cofactor) of XOR, eliminating NAD + binding. 3, 4 Once generated, these ROS can interfere with a multitude of cellular functions and processes. Two of the most extensively studied are the integrity of cellular membrane lipids and the interactions of superoxide with the vasodilator NO in circulation. In the latter case, superoxide from xanthine oxidase has been shown to degrade S-nitrothiols, which are considered to be a storage form of NO, as well as converting NO to the vaso-inactive compound peroxynitrite. 8,9 Superoxide has also been shown to reduce H 2 O 2 to form destructive hydroxyl radicals, and even carbonate radicals. 10 * To whom correspondence may be addressed T: (951) XOR is thus potentially a main player in many pathological conditions, and additio...

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...

Xanthine oxidase catalyzes the sequential hydroxylation of hypoxanthine to uric acid via xanthine as intermediate. Deposition of crystals of the catalytic product uric acid or its monosodium salt in human joints with accompanying joint inflammation is the major cause of gout. Natural flavonoids are attractive leads for rational design of preventive and therapeutic xanthine oxidase inhibitors due to their beneficial antioxidant, anti-inflammatory, and antiproliferative activities in addition to their micromolar inhibitory activities toward xanthine oxidase. We determined the first complex X-ray structure of mammalian xanthine oxidase with the natural flavonoid inhibitor quercetin at 2.0 Å resolution. The inhibitor adopts a single orientation with its benzopyran moiety sandwiched between Phe 914 and Phe 1009 and ring B pointing toward the solvent channel leading to the molybdenum active center. The favorable steric complementarity of the conjugated three-ring structure of quercetin with the active site and specific hydrogen-bonding interactions of exocyclic hydroxy groups with catalytically relevant residues Arg 880 and Glu 802 correlate well with a previously reported structure-activity relationship of flavonoid inhibitors of xanthine oxidase. The current complex provides a structural basis for the rational design of flavonoid-type inhibitors against xanthine oxidase useful for the treatment of hyperuricemia, gout, and inflammatory disease states.

Venetoclax (Ven) is a selective small‐molecule inhibitor of BCL‐2 that exhibits antitumoral activity against MM cells with t(11;14) translocation. We evaluated the safety and efficacy of Ven and dexamethasone (VenDex) combination in patients with t(11;14) positive relapsed/refractory (R/R) multiple myeloma (MM). This open‐label, multicenter study had two distinct phases (phase one [P1], phase two [P2]). Patients in both phases received VenDex (oral Ven 800 mg/day + oral Dex 40 mg [20 mg for patients ≥75 years] on days 1, 8, and 15, per 21–day cycle). The primary objective of the P1 VenDex cohort was to assess safety and pharmacokinetics. Phase two further evaluated efficacy with objective response rate (ORR) and very good partial response or better. Correlative studies explored baseline BCL2 (BCL‐2) and BCL2L1 (BCL‐XL) gene expression, cytogenetics, and recurrent somatic mutations in MM. Twenty and 31 patients in P1 and P2 with t(11;14) positive translocation received VenDex. P1/P2 patients had received a median of 3/5 lines of prior therapy, and 20%/87% were refractory to daratumumab. Predominant grade 3/4 hematological adverse events (AEs) with ≥10% occurrence included lymphopenia (20%/19%), neutropenia (15%/7%), thrombocytopenia (10%/10%), and anemia (5%/16%). At a median follow‐up of 12.3/9.2 months, ORR was 60%/48%. The duration of response estimate at 12 months was 50%/61%, and the median time to progression was 12.4/10.8 months. In biomarker evaluable patients, response to VenDex was independent of concurrent del(17p) or gain(1q) and mutations in key oncogenic signaling pathways, including MAPK and NF‐kB. VenDex demonstrated efficacy and manageable safety in heavily‐pre‐treated patients with t(11;14) R/R MM.

Ibrutinib use requires prudent consideration of the impacts on host immunity. We identified a high rate of serious adverse infectious events within prospective clinical trials. Data suggest a role of both BTK and ITK inhibition for the increased events. There was considerable variability in the reporting of adverse events between trials, journals, and conference reports.