xanthine oxidase

XANTHINE OXIDASE

(Prepared by Özgün Babur as homework of Biochemistry lecture)

Name: xanthine oxidase (recommended); hypoxanthine oxidase; xanthine:oxygen oxidoreductase (systematic name); hypoxanthine:oxygen oxidoreductase; Schardinger enzyme; xanthine oxidoreductase; hypoxanthine-xanthine oxidase; xanthine:O2 oxidoreductase; xanthine:xanthine oxidase

Ec no: 1.1.3.22

Sequence: XOHUDH . xanthine dehydroge...[gi:2144322]

aas: 1333 aa bps (mRNA): 3999 bp

In human genome:
It is composed of 36 exons and 35 introns and spans at least 60 kb. The exon sizes range from 53 to 279 bp, and the intron sizes range from 0.2 to more than 8 kb [1].

gene: XDH optimal pH: 8.2
Locus: 2p22.3-p22.2 (human)

Promoter structure:
Using primer extension and RNase protection analysis they identified 2 transcriptional initiation sites 59 and 82 nucleotides upstream of the ATG start codon. They found 1 Goldberg-Hogness box (ATTTAT)-like sequence 24 bp upstream from the second transcriptional initiation site and 2 inverted CCAAT sequences 19 and 42 bp upstream from the second transcriptional initiation site [1].

(my drawing as an illustration)

Reaction Catalysed:
Xanthine + H2O + O2 urate + H2O2
RH + H2O + 2 O2 ROH + 2 H+ + 2 O2-

figure taken from [2].

Hypoxanthine is oxidized successively to xanthine and then to uric acid by xanthine oxidase. Molecular oxygen is the electron acceptor in this complex reaction. Also oxidizes hypoxanthine, some other purines and pterins, and aldehydes [2].

Prosthetic groups:
One iron-sulphur center:

[3]

One molybdenum center:

[3]

Flavin adenine dinucleotide (FAD):

[3]

Structural domains, post-translational modifications:
It is a homodimer and contains one FAD, one molybdenum center and two iron-sulphur centers per monomer.

Xanthine dehydrogenase and xanthine oxidase are inter-convertible in the organism by reversible sulfhydryl oxidation or by irreversible proteolytic modification. These two enzymes are the protein product of the same gene.

Homologue protein Aldehyde Oxidase:
Aldehyde oxidase of Desulfovibrio Gigas is the only homologue protein of xanthine oxidase whose structure has been modelled by X-ray diffraction. It has 52% sequence identity with xanthine oxidase.

[4] [5] My drawing with Rasmol

Diseases Related:
Molybdenum Cofactor Deficiency (molybdenum cofactor is not functional, so XO can not work)
Xanthinuria, Type I (deficiency of xanthine dehydrogenase)
Xanthinuria, Type II (dual deficiency of xanthine dehydrogenase and aldehyde oxidase)
Hypouricemia, Hypercalcinuria, and Decreased Bone Density
--> All diseases above are because of insufficient functioning of xanthine oxidase/dehydrogenase. The similar symptoms are increased xanthine excretion, decreased uric acid excretion, mental retardation, etc [6].

Cancer is also one disease that is thought to be contributed by xanthine oxidase. One possible product of xanthine oxidase is superoxide radical (O2-), which is very carcinogen.

One proposed hypothesis says that drinking tea decreases the risk of cancer because the polypheols in it. Polyphenols are inhibitors of xanthine oxidase [7].

Mutations found:
C-to-T transition of nucleotide 682, caused a CGA (arg) to TGA (stop) nonsense change of codon 228. (xanthinuria type I) [8].
Deletion of nucleotide 2567C in XDH cDNA that generated a termination codon from nucleotide 2783. (xanthinuria type I) [8].

Some place that we can find xanthine oxidase:
Milk, Colostrum (foremilk), Liver, Duodenum, Pancreas, Kidney, Lung, Spleen, Cell, Seedlings, Cell culture (mouse embryo cell lines: 3T3, 3T6, B-3T3, 3T12)

Species specific expression:
It is found in a broad range of organisms (from bacteria to mammals).

It is detected in these organisms:
Bovine, Goat, Human, Sheep, Mouse, Rat, Dog, Patas monkey, Guinea pig, Rabbit, Donkey, Horse, Cat, Chicken, Drosophila melanogaster, Locust, Lens esculenta, Arthrobacter sp., Enterobacter cloacae.

Catalytic site:

Drawn by me taking reference [9]

Proposed mechanisms for the action of Mo center:

[10]

Xanthine and hypoxanthine are oxidized at the molybdenum center, the metal being reduced from the VI to the IV valence state; the reducing equivalents are transferred to molecular oxygen at the FAD with the mediation of the iron-sulfur centers [11].

When we scan its sequence with in PROSITE, we find:

3      N-glycosylation site
15    Protein kinase C phosphorylation site
19    Casein kinase II phosphorylation site
34    N-myristoylation site.
1      Amidation site
1      Eukaryotic molybdopterin oxidoreductases signature
1      2Fe-2S ferredoxins, iron-sulfur binding region signature

Regulation of its activity:
Inhibitors: Arsenite, Cyanide, Methanol, 2,4-Dinitrofluorobenzene, Formaldehyde, Urea, Guanine, Salicylate, Thiocyanate, 2,4-Diamino-6-hydroxy-s-triazine, 2-Amino--hydroxypterine-6-aldehyde, Purine-6-aldehyde, Allopurinol, Hydroxylamine, Semicarbazide, Phenylhydrazine, Myoglobin (inhibits reaction with cytochrome c as acceptor), Purines, Pterines (or other heterocyclic compounds, which are either not oxidized or oxidized rather slowly), 2-Amino-4-hydroxy-6-formylpterine, 3,3',4,4'-Tetrahydroxychalcone, Copper, p-Aminophenol quinimine, Dinitrophenol quinimine, 1,2-Dihydroxybenzene 3,5-disulfonic acid (inhibits reaction with cytochrome c), Ascorbic acid, Tetraethylthiuram disulfide, Borate, Imidazotriazines, Chalcones, Isatin, Ninhydrin, Alloxan, 8-Hydroxyquinoline-7-sulfonic acid, p-hloromercuribenzoate, Azaguanine, Aldehydes (e.g. formaldehyde, 4-yridinecarboxaldehyde, propionaldehyde, glycolaldehyde), Ag2+ , Cu2+ , Fe3+ , Co2+ , H2O2, Xanthine (substrate inhibition at high concentration).
Stabilizer: Phosphate

Superoxide generating system:
In order to produce superoxide radicals in vitro, we add xanthine and xanthine oxidase in the test tube.

References

1. Xu, P.; Zhu, X. L.; Huecksteadt, T. P.; Brothman, A. R.; Hoidal, J. R. : Assignment of human xanthine dehydrogenase gene to chromosome 2p22. Genomics23: 289-291, 1994.

2. Grossman, L. and K. Moldave, eds (1967) Methods in Enzymology, Vol XII, Nucleic Acids, Part A, Academic Press, NY, pg 5.

3. Degtyarenko, K.N., North, A.C.T. and Findlay, J.B.C. (1999) PROMISE: a database of bioinorganic motifs. Nucleic Acids Res. 27, 233-236.

4. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.

5. H.M.Berman, J.Westbrook, Z.Feng, G.Gilliland, T.N.Bhat, H.Weissig,I.N.Shindyalov, P.E.Bourne. (2000) The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242.

6. Online Mendelian Inheritance in Man, OMIM (TM). McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), 2000.

7. Ruch R.J. Cheng S. and Klaunig J.E. (1989) Carcinogenesis, 10, 1003-1008.

8. Ichida, K.; Amaya, Y.; Kamatani, N.; Nishino, T.; Hosoya, T.; Sakai, O. (1997) Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria. J. Clin. Invest. 99: 2391-2397.

9. Ilich, P., Hille, R. (1999) Mechanism of Formamide Hydroxylation Catalyzed by a Molybdenum-Dithiolene Complex: A Model for Xanthine Oxidase Reactivity. J. Phys. Chem. B, 103(25), 5406 -5412.

10. Xia, M., Dempski, R., Hille, R. (1999) The Reductive Half-reaction of Xanthine Oxidase. J. Biol. Chem. 224(6), 3323-3330.

11. Rastelli, G., Costantino, L., Albasini, A. (1997) A Model of the Interaction of Substrates and Inhibitors with Xanthine Oxidase. Am. Chem. Soc., 119(13), 3007 -3016.

For any comment contact directly with Özgün Babur.

1.	Xu, P.; Zhu, X. L.; Huecksteadt, T. P.; Brothman, A. R.; Hoidal, J. R. : Assignment of human xanthine dehydrogenase gene to chromosome 2p22. Genomics23: 289-291, 1994.
2.	Grossman, L. and K. Moldave, eds (1967) Methods in Enzymology, Vol XII, Nucleic Acids, Part A, Academic Press, NY, pg 5.
3.	Degtyarenko, K.N., North, A.C.T. and Findlay, J.B.C. (1999) PROMISE: a database of bioinorganic motifs. Nucleic Acids Res. 27, 233-236.
4.	Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.
5.	H.M.Berman, J.Westbrook, Z.Feng, G.Gilliland, T.N.Bhat, H.Weissig,I.N.Shindyalov, P.E.Bourne. (2000) The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242.
6.	Online Mendelian Inheritance in Man, OMIM (TM). McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), 2000.
7.	Ruch R.J. Cheng S. and Klaunig J.E. (1989) Carcinogenesis, 10, 1003-1008.
8.	Ichida, K.; Amaya, Y.; Kamatani, N.; Nishino, T.; Hosoya, T.; Sakai, O. (1997) Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria. J. Clin. Invest. 99: 2391-2397.
9.	Ilich, P., Hille, R. (1999) Mechanism of Formamide Hydroxylation Catalyzed by a Molybdenum-Dithiolene Complex: A Model for Xanthine Oxidase Reactivity. J. Phys. Chem. B, 103(25), 5406 -5412.
10.	Xia, M., Dempski, R., Hille, R. (1999) The Reductive Half-reaction of Xanthine Oxidase. J. Biol. Chem. 224(6), 3323-3330.
11.	Rastelli, G., Costantino, L., Albasini, A. (1997) A Model of the Interaction of Substrates and Inhibitors with Xanthine Oxidase. Am. Chem. Soc., 119(13), 3007 -3016.