Literature summary for 1.17.1.4 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 .

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
C535A/C992R	site-directed mutagenesis, the mutant activity in the presence of sulfhydryl residue modifiers is very low	Rattus norvegicus
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
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
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]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Molybdenum	in the molybdopterin cofactor	Rattus norvegicus

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Rattus norvegicus	P22985	-	-

Source Tissue

Source Tissue	Comment	Organism	Textmining
endothelial cell	-	Rattus norvegicus	-
liver	-	Rattus norvegicus	-

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

Synonyms	Comment	Organism
xanthine dehydrogenase/oxidase	UniProt	Rattus norvegicus
xanthine oxidoreductase	-	Rattus norvegicus
XDH	-	Rattus norvegicus
XOR	-	Rattus norvegicus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Rattus norvegicus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.8	-	assay at	Rattus norvegicus

Cofactor

Cofactor	Comment	Organism
FAD	one FAD per enzyme molecule	Rattus norvegicus
molybdopterin	one molybdopterin per enzyme molecule	Rattus norvegicus
NAD+	-	Rattus norvegicus
[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

General Information	Comment	Organism
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