Cloned (Comment) | Organism |
---|---|
Escherichia coli Sphi606 or Sphi609 cells transformed with pSelect-LdXPRT, pSelectldxprtE198D, pSelect-ldxprtE215D, pSelect-ldxprtE215Q and pSelectldxprtE198,215D | Leishmania donovani |
Protein Variants | Comment | Organism |
---|---|---|
E198D | increases the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity, converts the xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Can accommodate GMP in the active site | Leishmania donovani |
E198D/E215D | catalyzes robust guanine phosphoribosylation. Exhibits similar kinetic parameters in the presence of Mg2+ or Mn2+ as the wild-type. Substitution of Mg2+ with Mn2+ does not alter the hypoxanthine phosphoribosylation activity | Leishmania donovani |
E215D | increases the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity, converts the xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Can accommodate GMP in the active site | Leishmania donovani |
E215Q | can accommodate GMP in the active site | Leishmania donovani |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
GMP | inhibition of the xanthine phosphoribosylation | Leishmania donovani |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0021 | - |
hypoxanthine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0034 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
0.0049 | - |
guanine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0059 | - |
diphosphate | mutant E198D/E215D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.0073 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
0.0073 | - |
xanthine | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0078 | - |
xanthine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0078 | - |
IMP | mutant E198D/E215D, at 25°C, in TM buffer | Leishmania donovani | |
0.008 | - |
IMP | mutant E198D, at 25°C, in TM buffer | Leishmania donovani | |
0.008 | - |
IMP | mutant E215D, at 25°C, in TM buffer | Leishmania donovani | |
0.008 | - |
IMP | mutant E215Q, at 25°C, in TM buffer | Leishmania donovani | |
0.008 | - |
IMP | wild-type, at 25°C, in TM buffer | Leishmania donovani | |
0.0162 | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
0.0181 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
0.0199 | - |
xanthine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.023 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
0.0244 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
0.025 | - |
guanine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0251 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
0.028 | - |
diphosphate | mutant E215Q, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.0288 | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
0.035 | - |
xanthine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.0382 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
0.039 | - |
diphosphate | wild-type, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.045 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
0.074 | - |
diphosphate | mutant E198D/E215D, in the presence of 8 mM IMP | Leishmania donovani | |
0.0913 | - |
xanthine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.092 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | wild-type, in TM buffer | Leishmania donovani | |
0.094 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E215Q, in TM buffer | Leishmania donovani | |
0.1 | - |
guanine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.1 | - |
guanine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.115 | - |
diphosphate | mutant E215D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.129 | - |
hypoxanthine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.133 | - |
diphosphate | mutant E198D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.358 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E198D, in TM buffer | Leishmania donovani | |
0.396 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E215D, in TM buffer | Leishmania donovani | |
0.458 | - |
hypoxanthine | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.524 | - |
hypoxanthine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
0.533 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E198D/E215D, in TM buffer | Leishmania donovani | |
0.567 | - |
hypoxanthine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | maximal catalytic activity with 0.06 mM | Leishmania donovani | |
Mn2+ | maximal catalytic activity with 0.06 mM. Substitution of Mn2+ for Mg2+ results in ca. 2fold increase in the XPRT turnover rate without altering the xanthine Km value. Replacement of Mg2+ with Mn2+ increases the 5-phospho-alpha-D-ribose 1-diphosphate Km value ca. 2fold. XPRT-catalyzed hypoxanthine phosphoribosylation reveals that substitution of Mg2+ with Mn2+ results in a ca. 4fold increase in the hypoxanthine kcat value and ca. 3fold decrease in the hypoxanthine Km value | Leishmania donovani |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
27000 | - |
gel filtration | Leishmania donovani |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Leishmania donovani | - |
- |
- |
Purification (Comment) | Organism |
---|---|
by gel filtration, more than 95% pure | Leishmania donovani |
Storage Stability | Organism |
---|---|
-80°C, 10 mM Tris-HCl buffer, pH 7.5 | Leishmania donovani |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
5-phospho-alpha-D-ribose 1-diphosphate + guanine | - |
Leishmania donovani | GMP + diphosphate | - |
? | |
5-phospho-alpha-D-ribose 1-diphosphate + hypoxanthine | - |
Leishmania donovani | IMP + diphosphate | - |
r | |
5-phospho-alpha-D-ribose 1-diphosphate + xanthine | - |
Leishmania donovani | 9-(5-phospho-beta-D-ribosyl)xanthine + diphosphate | - |
? | |
9-(5-phospho-beta-D-ribosyl)xanthine + diphosphate | - |
Leishmania donovani | 5-phospho-alpha-D-ribose 1-diphosphate + xanthine | - |
? | |
IMP + diphosphate | - |
Leishmania donovani | 5-phospho-alpha-D-ribose 1-diphosphate + hypoxanthine | - |
r |
Synonyms | Comment | Organism |
---|---|---|
XPRT | - |
Leishmania donovani |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00367 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E215D, at 25°C, in TM buffer | Leishmania donovani | |
0.0038 | - |
diphosphate | mutant E215D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.0045 | - |
diphosphate | mutant E198D/E215D, in the presence of 8 mM IMP | Leishmania donovani | |
0.006167 | - |
IMP | mutant E198D/E215D, at 25°C, in TM buffer | Leishmania donovani | |
0.0093 | - |
diphosphate | mutant E198D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.0103 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E198D, at 25°C, in TM buffer | Leishmania donovani | |
0.0118 | - |
diphosphate | mutant E198D/E215D, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.0148 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E198D/E215D, at 25°C, in TM buffer | Leishmania donovani | |
0.0228 | - |
diphosphate | wild-type, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.025 | - |
diphosphate | mutant E215Q, in the presence of 1 mM 9-(5-phospho-beta-D-ribosyl)xanthine | Leishmania donovani | |
0.028 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | wild-type, at 25°C, in TM buffer | Leishmania donovani | |
0.043 | - |
9-(5-phospho-beta-D-ribosyl)xanthine | mutant E215Q, at 25°C, in TM buffer | Leishmania donovani | |
0.1 | - |
guanine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
1.1 | - |
guanine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
1.5 | - |
guanine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
2 | - |
xanthine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
2.1 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
2.2 | - |
hypoxanthine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
2.4 | - |
hypoxanthine | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
2.6 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215Q, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
3.1 | - |
hypoxanthine | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
3.3 | - |
guanine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
3.5 | - |
xanthine | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
3.6 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
3.9 | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
3.9 | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
6.7 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
7.1 | - |
hypoxanthine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
8.4 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM hypoxanthine | Leishmania donovani | |
8.7 | - |
hypoxanthine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
12.3 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
15.1 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
16.4 | - |
xanthine | mutant E198D/E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
17.5 | - |
xanthine | mutant E215D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani | |
20.7 | - |
5-phospho-alpha-D-ribose 1-diphosphate | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM xanthine | Leishmania donovani | |
23.3 | - |
xanthine | mutant E198D, at 25°C, 40 mM Tris-HCl, pH 7.5, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate | Leishmania donovani |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.039 | - |
GMP | wild-type | Leishmania donovani | |
0.044 | - |
GMP | mutant E215D | Leishmania donovani | |
0.067 | - |
GMP | mutant E198D/E215D | Leishmania donovani |
General Information | Comment | Organism |
---|---|---|
physiological function | Sphi609 cells expressing wild-type XPRT grow in minimal medium supplemented with xanthine but not in medium containing hypoxanthine or guanine. In contrast, Escherichia coli Sphi609 transformed with E198D or E215D exhibit vigorous growth with xanthine or hypoxanthine but notably less growth with guanine. Sphi609 transformants expressing E215Q or E198D/E215D proliferate robustly in xanthine, hypoxanthine, and guanine | Leishmania donovani |