Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes | Physcomitrium patens |
the gene is located on chromosome 2, expression of His-tagged wild-type and mutant enzymes | Zea mays |
Crystallization (Comment) | Organism |
---|---|
recombinant His-tagged wild-type enzyme, hanging drop avpour diffusion method, 30-35 mg/ml protein is mixed with an equal volume of a precipitant solution containing 0.1 M HEPES, pH 7.5, 100 mM sodium acetate, 10% w/v PEG 4000, and 10% ethylene glycol, X-ray diffraction structure determination and analysis at 3.35 A resolution, molecular replacement | Physcomitrium patens |
recombinant His-tagged wild-type enzyme, hanging drop avpour diffusion method, 35 mg/ml protein is mixed with an equal volume of reservori solution containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 20% w/v PEG 2000 monomethyl ether, X-ray diffraction structure determination and analysis at 2.49 A resolution, molecular replacement | Zea mays |
Protein Variants | Comment | Organism |
---|---|---|
D250A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
D252A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
D25A | site-directed mutagenesis, inactive mutant | Physcomitrium patens |
D8A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Zea mays |
E247A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
H245A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
H99A | site-directed mutagenesis, inactive mutant | Physcomitrium patens |
additional information | generation of a functional gene knockout of PpNRH1 using a gene-replacement vector. Phenotype and Growth of enzyme knockout mutant in medium with nucleosides as the sole nitrogen source, overview | Physcomitrium patens |
Y241A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
Y244A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
Y249A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
Y255A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Physcomitrium patens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | substrate specificity and kinetics, overview | Zea mays | |
0.078 | - |
Inosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens | |
0.094 | - |
Inosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
0.116 | - |
Xanthosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens | |
0.134 | - |
Inosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens | |
0.14 | - |
Xanthosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
0.171 | - |
Xanthosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens | |
0.201 | - |
Inosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
0.206 | - |
Xanthosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
0.232 | - |
Xanthosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
0.301 | - |
Inosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
0.356 | - |
Xanthosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
0.367 | - |
Xanthosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
0.396 | - |
Xanthosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
0.487 | - |
Xanthosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.596 | - |
Xanthosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens | |
0.63 | - |
Inosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.76 | - |
Inosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
1.09 | - |
Inosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
1.208 | - |
Inosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
1390 | - |
Inosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Physcomitrium patens | A9TXA6 | - |
- |
Physcomitrium patens Gransden 2004 | A9TXA6 | - |
- |
Zea mays | B6T563 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes by cobalt affinity chromatography and gel filtration | Physcomitrium patens |
recombinant His-tagged wild-type and mutant enzymes by cobalt affinity chromatography and gel filtration | Zea mays |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
inosine + H2O | best substrate | Zea mays | D-ribose + hypoxanthine | - |
? | |
inosine + H2O | 87% of the activity with xanthosine | Physcomitrium patens | D-ribose + hypoxanthine | - |
? | |
inosine + H2O | 87% of the activity with xanthosine | Physcomitrium patens Gransden 2004 | D-ribose + hypoxanthine | - |
? | |
additional information | poor activity with adenosine, uridine, and guanosine, no activity with cytokinin riboside, cytidine, and trans-zeatin riboside. Substrate docking into the active site, overview | Physcomitrium patens | ? | - |
? | |
additional information | poor activity with adenosine, uridine, guanosine, trans-zeatin riboside, and cytokinin riboside, almost no activity with cytidine. Substrate docking into the active site, overview | Zea mays | ? | - |
? | |
additional information | poor activity with adenosine, uridine, and guanosine, no activity with cytokinin riboside, cytidine, and trans-zeatin riboside. Substrate docking into the active site, overview | Physcomitrium patens Gransden 2004 | ? | - |
? | |
xanthosine + H2O | best substrate | Physcomitrium patens | D-ribose + xanthine | - |
? | |
xanthosine + H2O | 70% of the activity with inosine | Zea mays | D-ribose + xanthine | - |
? | |
xanthosine + H2O | best substrate | Physcomitrium patens Gransden 2004 | D-ribose + xanthine | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PpNRH1 | - |
Physcomitrium patens |
purine NRH | - |
Physcomitrium patens |
purine NRH | - |
Zea mays |
purine nucleoside N-ribohydrolase | - |
Physcomitrium patens |
purine nucleoside N-ribohydrolase | - |
Zea mays |
ZmNRH3 | - |
Zea mays |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Physcomitrium patens |
30 | - |
assay at | Zea mays |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.06 | - |
Inosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.19 | - |
Xanthosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.43 | - |
Xanthosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
0.47 | - |
Inosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
0.58 | - |
Xanthosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
0.7 | - |
Inosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens | |
1.1 | - |
Inosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
1.1 | - |
Xanthosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens | |
1.3 | - |
Xanthosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
1.4 | - |
Inosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
2.5 | - |
Xanthosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
3 | - |
Inosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
5.7 | - |
Inosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
6 | - |
Inosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens | |
6.2 | - |
Xanthosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
7 | - |
Xanthosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens | |
7.2 | - |
Inosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
7.2 | - |
Inosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens | |
7.2 | - |
Xanthosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
8.4 | - |
Xanthosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Physcomitrium patens |
7.5 | - |
assay at | Zea mays |
General Information | Comment | Organism |
---|---|---|
malfunction | changes in the levels of purine, pyrimidine, and cytokinin metabolites in knockout mutants, phenotypes, overview | Physcomitrium patens |
additional information | all plant nucleoside N-ribohydrolases exhibit a conserved sequence motif, DTDPGIDD, at the N-terminus, and the second Asp of the DTDPGIDD conserved motif functions as the active site base | Zea mays |
additional information | the presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides. All plant nucleoside N-ribohydrolases exhibit a conserved sequence motif, DTDPGIDD, at the N-terminus, and the second Asp of the DTDPGIDD conserved motif functions as the active site base | Physcomitrium patens |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.1 | - |
Inosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.4 | - |
Xanthosine | pH 7.5, 30°C, mutant Y241A | Physcomitrium patens | |
0.51 | - |
Inosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens | |
0.62 | - |
Inosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
1 | - |
Inosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
1.2 | - |
Inosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
1.2 | - |
Xanthosine | pH 7.5, 30°C, mutant Y255A | Physcomitrium patens | |
1.8 | - |
Xanthosine | pH 7.5, 30°C, mutant Y249A | Physcomitrium patens | |
2.8 | - |
Xanthosine | pH 7.5, 30°C, mutant Y244A | Physcomitrium patens | |
3.6 | - |
Xanthosine | pH 7.5, 30°C, mutant H245A | Physcomitrium patens | |
10 | - |
Inosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
11 | - |
Xanthosine | pH 7.5, 30°C, mutant D252A | Physcomitrium patens | |
16 | - |
Xanthosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
36 | - |
Inosine | pH 7.5, 30°C, recombinant enzyme | Zea mays | |
49 | - |
Xanthosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens | |
51 | - |
Xanthosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
53 | - |
Inosine | pH 7.5, 30°C, mutant D250A | Physcomitrium patens | |
61 | - |
Xanthosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens | |
61 | - |
Inosine | pH 7.5, 30°C, mutant E247A | Physcomitrium patens | |
77 | - |
Inosine | pH 7.5, 30°C, wild-type enzyme | Physcomitrium patens |