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Literature summary 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)

EC Number Cloned (Comment) Organism
3.2.2.1 recombinant expression of His-tagged wild-type and mutant enzymes Physcomitrium patens
3.2.2.1 the gene is located on chromosome 2, expression of His-tagged wild-type and mutant enzymes Zea mays
3.2.2.3 isozyme PpNRH2, phylogenetic analysis, recombinant expression in Escherichia coli in inclusion bodies Physcomitrium patens
3.2.2.3 phylogenetic analysis Zea mays

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.2.2.1 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
3.2.2.1 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

EC Number Protein Variants Comment Organism
3.2.2.1 D250A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 D252A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 D25A site-directed mutagenesis, inactive mutant Physcomitrium patens
3.2.2.1 D8A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Zea mays
3.2.2.1 E247A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 H245A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 H99A site-directed mutagenesis, inactive mutant Physcomitrium patens
3.2.2.1 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
3.2.2.1 Y241A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 Y244A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 Y249A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.1 Y255A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Physcomitrium patens
3.2.2.3 additional information functional knockout of the mutant, phenotypic analysis of wild-type and mutant enzymes, overview Physcomitrium patens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.2.1 additional information
-
 additional information substrate specificity and kinetics, overview Zea mays
3.2.2.1 0.078
-
 Inosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.1 0.094
-
 Inosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 0.116
-
 Xanthosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.1 0.134
-
 Inosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.1 0.14
-
 Xanthosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 0.171
-
 Xanthosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.1 0.201
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 0.206
-
 Xanthosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 0.232
-
 Xanthosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 0.301
-
 Inosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 0.356
-
 Xanthosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 0.367
-
 Xanthosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 0.396
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 0.487
-
 Xanthosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.596
-
 Xanthosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.1 0.63
-
 Inosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.76
-
 Inosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 1.09
-
 Inosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 1.208
-
 Inosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 1390
-
 Inosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.3 additional information
-
 additional information substrate specificity and kinetics, overview Zea mays
3.2.2.3 0.06
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.109
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.111
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.178
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.468
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.512
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.713
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 1.013
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.2.2.3 uridine + H2O Zea mays
-
 D-ribose + uracil
-
 ?
3.2.2.3 uridine + H2O Physcomitrium patens
-
 D-ribose + uracil
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.2.2.1 Physcomitrium patens A9TXA6
-
-
3.2.2.1 Physcomitrium patens Gransden 2004 A9TXA6
-
-
3.2.2.1 Zea mays B6T563
-
-
3.2.2.3 Physcomitrium patens
-
-
-
3.2.2.3 Zea mays
-
-
-
3.2.2.3 Zea mays H9D3U7
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.2.1 recombinant His-tagged wild-type and mutant enzymes by cobalt affinity chromatography and gel filtration Physcomitrium patens
3.2.2.1 recombinant His-tagged wild-type and mutant enzymes by cobalt affinity chromatography and gel filtration Zea mays

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.2.1 inosine + H2O best substrate Zea mays D-ribose + hypoxanthine
-
 ?
3.2.2.1 inosine + H2O 87% of the activity with xanthosine Physcomitrium patens D-ribose + hypoxanthine
-
 ?
3.2.2.1 inosine + H2O 87% of the activity with xanthosine Physcomitrium patens Gransden 2004 D-ribose + hypoxanthine
-
 ?
3.2.2.1 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 ?
-
 ?
3.2.2.1 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 ?
-
 ?
3.2.2.1 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 ?
-
 ?
3.2.2.1 xanthosine + H2O best substrate Physcomitrium patens D-ribose + xanthine
-
 ?
3.2.2.1 xanthosine + H2O 70% of the activity with inosine Zea mays D-ribose + xanthine
-
 ?
3.2.2.1 xanthosine + H2O best substrate Physcomitrium patens Gransden 2004 D-ribose + xanthine
-
 ?
3.2.2.3 adenosine + H2O 15% of the activity with uridine Zea mays D-ribose + adenine
-
 ?
3.2.2.3 adenosine + H2O 3.5% of the activity with uridine Zea mays D-ribose + adenine
-
 ?
3.2.2.3 cytidine + H2O 3.9% of the activity with uridine Physcomitrium patens D-ribose + cytosine
-
 ?
3.2.2.3 cytidine + H2O 5.0% of the activity with uridine Zea mays D-ribose + cytosine
-
 ?
3.2.2.3 inosine + H2O 22% of the activity with uridine Physcomitrium patens D-ribose + hypoxanthine
-
 ?
3.2.2.3 inosine + H2O 9.3% of the activity with uridine Zea mays D-ribose + hypoxanthine
-
 ?
3.2.2.3 inosine + H2O 9.5% of the activity with uridine Zea mays D-ribose + hypoxanthine
-
 ?
3.2.2.3 additional information adenosine is a poor substrates, no activity with guanosine, trans-zeatin riboside, and cytokinin riboside, substrate specificity, overview Physcomitrium patens ?
-
 ?
3.2.2.3 additional information no or almost no activity with guanosine, trans-zeatin riboside, and cytokinin riboside, substrate specificity, overview Zea mays ?
-
 ?
3.2.2.3 additional information poor activity with cytidine, guanosine, trans-zeatin riboside, and cytokinin riboside, substrate specificity, overview Zea mays ?
-
 ?
3.2.2.3 uridine + H2O
-
 Zea mays D-ribose + uracil
-
 ?
3.2.2.3 uridine + H2O
-
 Physcomitrium patens D-ribose + uracil
-
 ?
3.2.2.3 uridine + H2O best substrate Zea mays D-ribose + uracil
-
 ?
3.2.2.3 uridine + H2O best substrate Physcomitrium patens D-ribose + uracil
-
 ?
3.2.2.3 xanthosine + H2O 34% of the activity with uridine Zea mays D-ribose + xanthine
-
 ?
3.2.2.3 xanthosine + H2O 53% of the activity with uridine Zea mays D-ribose + xanthine
-
 ?
3.2.2.3 xanthosine + H2O 61% of the activity with uridine Physcomitrium patens D-ribose + xanthine
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.2.1 PpNRH1
-
 Physcomitrium patens
3.2.2.1 purine NRH
-
 Physcomitrium patens
3.2.2.1 purine NRH
-
 Zea mays
3.2.2.1 purine nucleoside N-ribohydrolase
-
 Physcomitrium patens
3.2.2.1 purine nucleoside N-ribohydrolase
-
 Zea mays
3.2.2.1 ZmNRH3
-
 Zea mays
3.2.2.3 PpNRH2
-
 Physcomitrium patens
3.2.2.3 ZmNRH2a
-
 Zea mays
3.2.2.3 ZmNRH2b
-
 Zea mays

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.2.1 30
-
 assay at Physcomitrium patens
3.2.2.1 30
-
 assay at Zea mays
3.2.2.3 30
-
 assay at Zea mays
3.2.2.3 30
-
 assay at Physcomitrium patens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.2.1 0.06
-
 Inosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.19
-
 Xanthosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.43
-
 Xanthosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 0.47
-
 Inosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 0.58
-
 Xanthosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 0.7
-
 Inosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.1 1.1
-
 Inosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 1.1
-
 Xanthosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.1 1.3
-
 Xanthosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 1.4
-
 Inosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 2.5
-
 Xanthosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 3
-
 Inosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 5.7
-
 Inosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 6
-
 Inosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.1 6.2
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 7
-
 Xanthosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.1 7.2
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 7.2
-
 Inosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.1 7.2
-
 Xanthosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 8.4
-
 Xanthosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.3 0.18
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.4
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 0.61
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 1.3
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 3
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 4.1
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 4.6
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 23.8
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.2.1 7.5
-
 assay at Physcomitrium patens
3.2.2.1 7.5
-
 assay at Zea mays
3.2.2.3 7.5
-
 assay at Zea mays
3.2.2.3 7.5
-
 assay at Physcomitrium patens

General Information

EC Number General Information Comment Organism
3.2.2.1 malfunction changes in the levels of purine, pyrimidine, and cytokinin metabolites in knockout mutants, phenotypes, overview Physcomitrium patens
3.2.2.1 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
3.2.2.1 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
3.2.2.3 malfunction changes in the levels of purine, pyrimidine, and cytokinin metabolites in knockout mutants, phenotypes, overview Physcomitrium patens
3.2.2.3 additional information the presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides Zea mays
3.2.2.3 additional information the presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides Physcomitrium patens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.2.1 0.1
-
 Inosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.4
-
 Xanthosine pH 7.5, 30°C, mutant Y241A Physcomitrium patens
3.2.2.1 0.51
-
 Inosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.1 0.62
-
 Inosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 1
-
 Inosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 1.2
-
 Inosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 1.2
-
 Xanthosine pH 7.5, 30°C, mutant Y255A Physcomitrium patens
3.2.2.1 1.8
-
 Xanthosine pH 7.5, 30°C, mutant Y249A Physcomitrium patens
3.2.2.1 2.8
-
 Xanthosine pH 7.5, 30°C, mutant Y244A Physcomitrium patens
3.2.2.1 3.6
-
 Xanthosine pH 7.5, 30°C, mutant H245A Physcomitrium patens
3.2.2.1 10
-
 Inosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 11
-
 Xanthosine pH 7.5, 30°C, mutant D252A Physcomitrium patens
3.2.2.1 16
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 36
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.1 49
-
 Xanthosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.1 51
-
 Xanthosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 53
-
 Inosine pH 7.5, 30°C, mutant D250A Physcomitrium patens
3.2.2.1 61
-
 Xanthosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.1 61
-
 Inosine pH 7.5, 30°C, mutant E247A Physcomitrium patens
3.2.2.1 77
-
 Inosine pH 7.5, 30°C, wild-type enzyme Physcomitrium patens
3.2.2.3 0.6
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 3
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 3.5
-
 adenosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 5.7
-
 Inosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 6.4
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 7.5
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 42
-
 Xanthosine pH 7.5, 30°C, recombinant enzyme Zea mays
3.2.2.3 46
-
 uridine pH 7.5, 30°C, recombinant enzyme Zea mays