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Literature summary extracted from

  • Nishino, T.; Okamoto, K.; Kawaguchi, Y.; Matsumura, T.; Eger, B.T.; Pai, E.F.; Nishino, T.
    The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase (2015), FEBS J., 282, 3075-3090 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.17.1.4 purified recombinant C-terminally truncated mutant enzyme, crystals of the mutant protein are prepared in two ways: (a) crystallization of the protein directly after DTT treatment and (b) crystallization in the presence of DTT followed by extended soaks in mother liquor devoid of DTT to convert most of the protein to the XO form, X-ray diffraction structure determination and analysis at 2.0 A resolution. Comparisons of crystal structures of a stable wild-type XDH enzyme form, the triple mutant C535A/C992R/C1324, and the DELTAC truncated mutant XOR Rattus norvegicus
1.17.3.2 purified recombinant C-terminally truncated mutant enzyme, crystals of the mutant protein are prepared in two ways: (a) crystallization of the protein directly after DTT treatment and (b) crystallization in the presence of DTT followed by extended soaks in mother liquor devoid of DTT to convert most of the protein to the XO form, X-ray diffraction structure determination and analysis at 2.0 A resolution. Comparisons of crystal structures of a stable wild-type XDH enzyme form, the triple mutant C535A/C992R/C1324, and the DELTAC truncated mutant XOR Rattus norvegicus

Protein Variants

EC Number Protein Variants Comment Organism
1.17.1.4 C535A/C992R site-directed mutagenesis, the mutant activity in the presence of sulfhydryl residue modifiers is very low Rattus norvegicus
1.17.1.4 C535A/C992R/C1316S site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all Rattus norvegicus
1.17.1.4 C535A/C992R/C1324S site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all Rattus norvegicus
1.17.1.4 additional information construction of a variant of the rat liver enzyme that lacks the C-terminal amino acids 1316-1331. The mutant enzymes appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified mutant protein retains about 50-70% of oxidase activity even after prolonged dithiothreitol treatment. 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. But after 15 min of incubation with a high concentration of NADH, the corresponding X-ray structures show 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 mutant 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 Rattus norvegicus
1.17.3.2 C535A/C992R site-directed mutagenesis, the mutant activity in the presence of sulfhydryl residue modifiers is very low Rattus norvegicus
1.17.3.2 C535A/C992R/C1316S site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all Rattus norvegicus
1.17.3.2 C535A/C992R/C1324S site-directed mutagenesis, the triple mutant does not undergo conversion from XOR, EC 1.17.3.2, to XDH, EC 1.17.1.4, at all Rattus norvegicus
1.17.3.2 additional information construction of a variant of the rat liver enzyme that lacks the C-terminal amino acids 1316-1331. The mutant enzymes appears to assume an intermediate form, exhibiting a mixture of dehydrogenase and oxidase activities. The purified mutant protein retains about 50-70% of oxidase activity even after prolonged dithiothreitol treatment. 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. But after 15 min of incubation with a high concentration of NADH, the corresponding X-ray structures show 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 mutant 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 Rattus norvegicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.17.1.4 additional information
-
additional information steady-state kinetics of DTT-treated and untreated C-terminally truncated enzyme mutant Rattus norvegicus
1.17.1.4 0.0052
-
NAD+ DTT-treated C-terminally truncated enzyme mutant, pH 7.8, 25°C Rattus norvegicus
1.17.3.2 additional information
-
additional information steady-state kinetics of DTT-treated and untreated C-terminally truncated enzyme mutant Rattus norvegicus
1.17.3.2 0.0497
-
O2 untreated C-terminally truncated enzyme mutant, pH 7.8, 25°C Rattus norvegicus
1.17.3.2 0.0537
-
O2 DTT-treated C-terminally truncated enzyme mutant, pH 7.8, 25°C Rattus norvegicus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.17.1.4 Molybdenum in the molybdopterin cofactor Rattus norvegicus
1.17.3.2 Molybdenum in the molybdopterin cofactor Rattus norvegicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.17.1.4 xanthine + NAD+ + H2O Rattus norvegicus
-
urate + NADH + H+
-
?
1.17.3.2 xanthine + H2O + O2 Rattus norvegicus
-
urate + H2O2
-
?

Organism

EC Number Organism UniProt Comment Textmining
1.17.1.4 Rattus norvegicus P22985
-
-
1.17.3.2 Rattus norvegicus P22985
-
-

Source Tissue

EC Number Source Tissue Comment Organism Textmining
1.17.1.4 endothelial cell
-
Rattus norvegicus
-
1.17.1.4 liver
-
Rattus norvegicus
-
1.17.3.2 endothelial cell
-
Rattus norvegicus
-
1.17.3.2 liver
-
Rattus norvegicus
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.17.1.4 additional information purified recombinant wild-type and DELTAC mutant enzymes both exhibit mostly xanthine oxidase activity Rattus norvegicus ?
-
?
1.17.1.4 xanthine + NAD+ + H2O
-
Rattus norvegicus urate + NADH + H+
-
?
1.17.3.2 additional information purified recombinant wild-type and DELTAC mutant enzymes both exhibit mostly xanthine oxidase activity Rattus norvegicus ?
-
?
1.17.3.2 xanthine + H2O + O2
-
Rattus norvegicus urate + H2O2
-
?

Subunits

EC Number Subunits Comment Organism
1.17.1.4 ? x * 150000, about, C-terminally truncated mutant enzyme DELTAC, SDS-PAGE Rattus norvegicus
1.17.1.4 More enzyme structure analysis, overview Rattus norvegicus
1.17.3.2 ? x * 150000, about, C-terminally truncated mutant enzyme DELTAC, SDS-PAGE Rattus norvegicus
1.17.3.2 More enzyme structure analysis, overview Rattus norvegicus

Synonyms

EC Number Synonyms Comment Organism
1.17.1.4 xanthine dehydrogenase/oxidase UniProt Rattus norvegicus
1.17.1.4 xanthine oxidoreductase
-
Rattus norvegicus
1.17.1.4 XDH
-
Rattus norvegicus
1.17.1.4 XOR
-
Rattus norvegicus
1.17.3.2 xanthine dehydrogenase/oxidase UniProt Rattus norvegicus
1.17.3.2 xanthine oxidoreductase
-
Rattus norvegicus
1.17.3.2 XOR
-
Rattus norvegicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.17.1.4 25
-
assay at Rattus norvegicus
1.17.3.2 25
-
assay at Rattus norvegicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.17.1.4 7.8
-
assay at Rattus norvegicus
1.17.3.2 7.8
-
assay at Rattus norvegicus

Cofactor

EC Number Cofactor Comment Organism Structure
1.17.1.4 FAD one FAD per enzyme molecule Rattus norvegicus
1.17.1.4 molybdopterin one molybdopterin per enzyme molecule Rattus norvegicus
1.17.1.4 NAD+
-
Rattus norvegicus
1.17.1.4 [2Fe-2S]-center two nonidentical [2Fe-2S] clusters designated as Fe/SI and Fe/SII, distinguished by redox potential and EPR signal Rattus norvegicus
1.17.3.2 FAD one FAD per enzyme molecule Rattus norvegicus
1.17.3.2 molybdopterin one molybdopterin per enzyme molecule Rattus norvegicus
1.17.3.2 [2Fe-2S]-center two nonidentical [2Fe-2S] clusters designated as Fe/SI and Fe/SII, distinguished by redox potential and EPR signal Rattus norvegicus

General Information

EC Number General Information Comment Organism
1.17.1.4 physiological function mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase. Conversion between the two is implicated in such diverse processes as lactation, anti-bacterial activity, reperfusion injury and a growing number of diseases. 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. 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. Residues Cys535 and Cys992 are involved in the rapid phase and Cys1316 and Cys1324 in the slow phase of the modification reaction. The irreversible conversion of XDH to XOR by trypsin involves limited proteolysis at the same linker peptide. Triggering events, such as the formation of a disulfide bond between Cys535 and Cys992 or proteolysis of the linker, reorient Phe549 (also a part of the long linker), resulting in disruption of a four amino acid cluster. Arg426 is then released from the cluster and moves the A-loop that blocks the approach of NAD+ to the flavin ring of the FAD moiety, as well as changing the electrostatic environment Rattus norvegicus
1.17.3.2 physiological function mammalian xanthine oxidoreductase can exist in both dehydrogenase and oxidase forms. The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase. Conversion between the two is implicated in such diverse processes as lactation, anti-bacterial activity, reperfusion injury and a growing number of diseases. 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. 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. Residues Cys535 and Cys992 are involved in the rapid phase and Cys1316 and Cys1324 in the slow phase of the modification reaction. The irreversible conversion of XDH to XOR by trypsin involves limited proteolysis at the same linker peptide. Triggering events, such as the formation of a disulfide bond between Cys535 and Cys992 or proteolysis of the linker, reorient Phe549 (also a part of the long linker), resulting in disruption of a four amino acid cluster. Arg426 is then released from the cluster and moves the A-loop that blocks the approach of NAD+ to the flavin ring of the FAD moiety, as well as changing the electrostatic environment Rattus norvegicus