Intermediate dehydrogenase-oxidase form of xanthine oxidoreductase in rat liver.
Ontology highlight
ABSTRACT:
SUBMITTER: Kaminski ZW
PROVIDER: S-EPMC1161139 | biostudies-other | 1979 Jul
REPOSITORIES: biostudies-other
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Project description:UnlabelledMammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. Conversion between the two is implicated in such diverse processes as lactation, anti-bacterial activity, reperfusion injury and a growing number of diseases. We have constructed a variant of the rat liver enzyme that lacks the carboxy-terminal amino acids 1316-1331; it appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified variant protein retained ~ 50-70% of oxidase activity even after prolonged dithiothreitol treatment, supporting a previous prediction that the C-terminal region plays a role in the dehydrogenase to oxidase conversion. In the crystal structure of the protein variant, most of the enzyme stays in an oxidase conformation. After 15 min of incubation with a high concentration of NADH, however, the corresponding X-ray structures showed a dehydrogenase-type conformation. On the other hand, disulfide formation between Cys535 and Cys992, which can clearly be seen in the electron density map of the crystal structure of the variant after removal of dithiothreitol, goes in parallel with the complete conversion to oxidase, resulting in structural changes identical to those observed upon proteolytic cleavage of the linker peptide. These results indicate that the dehydrogenase-oxidase transformation occurs rather readily and the insertion of the C-terminal peptide into the active site cavity of its subunit stabilizes the dehydrogenase form. We propose that the intermediate form can be generated (e.g. in endothelial cells) upon interaction of the C-terminal peptide portion of the enzyme with other proteins or the cell membrane.DatabaseCoordinate sets and structure factors for the four crystal structures reported in the present study have been deposited in the Protein Data Bank under the identification numbers 4YRW, 4YTZ, 4YSW, and 4YTY.
| S-EPMC4832347 | biostudies-literature
Project description:Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth.
| S-EPMC6817904 | biostudies-literature
Project description:Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD(+)), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD(+) in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC.
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Project description:Inflammation and ischemia--reperfusion tissue injury are important pathophysiologic processes with a wide spectrum of clinical presentations; the enzyme xanthine dehydrogenase/oxidase (XDH/XO) is thought to play a key role in ischemia--reperfusion injury. Recent studies have shown the transcriptional regulation of XDH/XO by cytokines (Dupont et al., 1992, J. Clin. Invest. 89, 197-202). In the present study, the 5' structure of the XDH/XO gene and characterization of its promoter are undertaken providing an initial step to further elucidate the regulatory mechanism(s) of this enzyme. XDH/XO cDNA from rat bone marrow macrophage has been isolated and used to screen a rat genomic library in order to identify and characterize the promoter of the XDH/XO gene. By Southern analysis, XDH/XO was found to be a single copy gene in the rat genome. Primer extension, RNase protection, and anchor-PCR studies indicate the presence of multiple start sites within a 65 bp window located some 20-85 bp upstream of the translation initiator (ATG). Functional studies of the sequences up to 116 nt upstream of the translational start site, which encompasses the several transcriptional start sites, indicate that this region is sufficient to drive the expression of a luciferase reporter gene and is presumed to represent the promoter. Neither a TATA box nor a GC-rich region are present in close proximity to any of the transcriptional start sites; however, sequences with homology to known initiator elements are found within this 116 bp fragment. Several possible regulatory elements, including a NF-IL6 motif, are also located upstream of the transcriptional start site. This study represents the first description of the XDH/XO promoter from a vertebrate system.
| S-EPMC308083 | biostudies-other
Project description:Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (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-A resolution and XO at 2.5-A resolution and describe 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 containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.
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Project description:The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.
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Project description:Nitric oxide radical (NO) is a signaling molecule involved in several physiological and pathological processes and a new nitrate-nitrite-NO pathway has emerged as a physiological alternative to the "classic" pathway of NO formation from L-arginine. Since the late 1990s, it has become clear that nitrite can be reduced back to NO under hypoxic/anoxic conditions and exert a significant cytoprotective action in vivo under challenging conditions. To reduce nitrite to NO, mammalian cells can use different metalloproteins that are present in cells to perform other functions, including several heme proteins and molybdoenzymes, comprising what we denominated as the "non-dedicated nitrite reductases". Herein, we will review the current knowledge on two of those "non-dedicated nitrite reductases", the molybdoenzymes xanthine oxidoreductase and aldehyde oxidase, discussing the in vitro and in vivo studies to provide the current picture of the role of these enzymes on the NO metabolism in humans.
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Project description:When investigating the potential for xanthine oxidase (XO)-mediated metabolism of a new chemical entity in vitro, selective chemical inhibition experiments are typically used. Most commonly, these inhibition experiments are performed using the inhibitor allopurinol (AP) and commercially prepared human liver cytosol (HLC) as the enzyme source. For reasons detailed herein, it is also a common practice to perfuse livers with solutions containing AP prior to liver harvest. The exposure to AP in HLC preparations could obviously pose a problem for measuring in vitro XO activity. To investigate this potential problem, an HPLC-MS/MS assay was developed to determine whether AP and its primary metabolite, oxypurinol, are retained within the cytosol for livers that were treated with AP during liver harvest. Differences in enzymatic activity for XO and aldehyde oxidase (AO) in human cytosol that can be ascribed to AP exposure were also evaluated. The results confirmed the presence of residual AP (some) and oxypurinol (all) human liver cytosol preparations that had been perfused with an AP-containing solution. In every case where oxypurinol was detected, XO activity was not observed. In contrast, the presence of AP and oxypurinol did not appear to have an impact on AO activity. Pooled HLC that was purchased from a commercial source also contained residual oxypurinol and did not show any XO activity. In the future, it is recommended that each HLC batch is screened for oxypurinol and/or XO activity prior to testing for XO-mediated metabolism of a new chemical entity.
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Project description:We hypothesized that XDH plays a role in nephrogenesis. Here we used SSXdh+/+, SSXdh+/-, and SSXdh-/- rat kidneys from the postnatal development period to test this hypothesis. In wildtype but not homozygote rat kidneys, XDH expression was significantly increased from postnatal day (PND) 5 to 15, indicating its importance in the maturation of the kidney. Even though both genotypes had similar glomerular density at birth, it was significantly lower in SSXdh-/- rats by PND21. Transcriptomic profiling of the kidneys revealed that many genes related to kidney formation were dysregulated by the lack of Xdh expression in this period. Functional and pathway analyses of the transcriptome data strongly suggested that Xdh affected these genes via epidermal growth factor (EGF) signaling disruption. This study showed that Xdh is essential for the kidney maturation process.
2023-09-20 | GSE239437 | GEO
Project description:The senescence process is the result of a series of factors that start from the genetic constitution interacting with epigenetic modifications induced by endogenous and environmental causes and that lead to a progressive deterioration at the cellular and functional levels. One of the main causes of aging is oxidative stress deriving from the imbalance between the production of reactive oxygen (ROS) and nitrogen (RNS) species and their scavenging through antioxidants. Xanthine oxidoreductase (XOR) activities produce uric acid, as well as reactive oxygen and nitrogen species, which all may be relevant to such equilibrium. This review analyzes XOR activity through in vitro experiments, animal studies and clinical reports, which highlight the pro-aging effects of XOR products. However, XOR activity contributes to a regular level of ROS and RNS, which appears essential for the proper functioning of many physiological pathways. This discourages the use of therapies with XOR inhibitors, unless symptomatic hyperuricemia is present.
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