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Literature summary for 3.2.2.1 extracted from

  • Kopecna, M.; Blaschke, H.; Kopecny, D.; Vigouroux, A.; Koncitikova, R.; Novak, O.; Kotland, O.; Strnad, M.; Morera, S.; von Schwartzenberg, K.
    Structure and function of nucleoside hydrolases from Physcomitrella patens and maize catalyzing the hydrolysis of purine, pyrimidine, and cytokinin ribosides (2013), Plant Physiol., 163, 1568-1583.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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 [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

Organism UniProt Comment Textmining
Physcomitrium patens A9TXA6
-
-
Physcomitrium patens Gransden 2004 A9TXA6
-
-
Zea mays B6T563
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Physcomitrium patens
30
-
assay at Zea mays

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Physcomitrium patens
7.5
-
assay at Zea mays

General Information

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 [mM/s]

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