BRENDA - Enzyme Database

The C-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase

Nishino, T.; Okamoto, K.; Kawaguchi, Y.; Matsumura, T.; Eger, B.T.; Pai, E.F.; Nishino, T.; FEBS J. 282, 3075-3090 (2015)

Data extracted from this reference:

Crystallization (Commentary)
EC Number
Crystallization
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
Engineering
EC Number
Amino acid exchange
Commentary
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
Commentary
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
Commentary
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
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
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
Primary Accession No. (UniProt)
Commentary
Textmining
1.17.1.4
Rattus norvegicus
P22985
-
-
1.17.3.2
Rattus norvegicus
P22985
-
-
Source Tissue
EC Number
Source Tissue
Commentary
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
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.17.1.4
additional information
purified recombinant wild-type and DELTAC mutant enzymes both exhibit mostly xanthine oxidase activity
744875
Rattus norvegicus
?
-
-
-
-
1.17.1.4
xanthine + NAD+ + H2O
-
744875
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
744875
Rattus norvegicus
?
-
-
-
-
1.17.3.2
xanthine + H2O + O2
-
744875
Rattus norvegicus
urate + H2O2
-
-
-
?
Subunits
EC Number
Subunits
Commentary
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
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
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
Commentary
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
Commentary
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
Cofactor (protein specific)
EC Number
Cofactor
Commentary
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
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
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
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
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] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
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 (protein specific)
EC Number
Metals/Ions
Commentary
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) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.17.1.4
xanthine + NAD+ + H2O
Rattus norvegicus
-
urate + NADH + H+
-
-
?
1.17.3.2
xanthine + H2O + O2
Rattus norvegicus
-
urate + H2O2
-
-
?
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
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) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.17.1.4
additional information
purified recombinant wild-type and DELTAC mutant enzymes both exhibit mostly xanthine oxidase activity
744875
Rattus norvegicus
?
-
-
-
-
1.17.1.4
xanthine + NAD+ + H2O
-
744875
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
744875
Rattus norvegicus
?
-
-
-
-
1.17.3.2
xanthine + H2O + O2
-
744875
Rattus norvegicus
urate + H2O2
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
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
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.17.1.4
25
-
assay at
Rattus norvegicus
1.17.3.2
25
-
assay at
Rattus norvegicus
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.17.1.4
7.8
-
assay at
Rattus norvegicus
1.17.3.2
7.8
-
assay at
Rattus norvegicus
General Information
EC Number
General Information
Commentary
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
General Information (protein specific)
EC Number
General Information
Commentary
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