Information on EC 3.2.2.8 - ribosylpyrimidine nucleosidase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea

EC NUMBER
COMMENTARY hide
3.2.2.8
-
RECOMMENDED NAME
GeneOntology No.
ribosylpyrimidine nucleosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a pyrimidine nucleoside + H2O = D-ribose + a pyrimidine base
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of N-glycosyl bond
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
Purine metabolism
-
-
Pyrimidine metabolism
-
-
pyrimidine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
pyrimidine-nucleoside ribohydrolase
Also hydrolyses purine D-ribonucleosides, but much more slowly. 2'-, 3'- and 5'-deoxynucleosides are not substrates [3].
CAS REGISTRY NUMBER
COMMENTARY hide
37288-60-1
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
a knockout mutant for NSH1 shows symptoms of accelerated senescence, accompanied by marked accumulation of uridine and xanthosine under conditions of prolonged darkness.
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-beta-D-ribofuranosylnicotinamide + H2O
nicotinamide + D-ribose
show the reaction diagram
-
-
-
?
1-beta-D-ribofuranosylthymine + H2O
D-ribose + thymine
show the reaction diagram
-
-
-
?
4-amino-5-imidazolecarboxamide ribonucleotide + H2O
4-amino-5-imidazolecarboxamide + D-ribose
show the reaction diagram
-
-
-
?
4-nitrophenyl ribopyranoside + H2O
4-nitrophenol + D-ribose
show the reaction diagram
-
-
-
?
5-bromouridine + H2O
5-bromouracil + D-ribose
show the reaction diagram
5-fluorouridine + H2O
5-fluorouracil + D-ribose
show the reaction diagram
5-iodouridine + H2O
5-iodouracil + D-ribose
show the reaction diagram
-
-
-
-
?
5-methyluridine + H2O
5-methyluracil + D-ribose
show the reaction diagram
-
-
-
-
?
5-methyluridine + H2O
D-ribose + 5-methyluracil
show the reaction diagram
-
-
-
-
?
6-azauridine + H2O
6-azauracil + D-ribose
show the reaction diagram
-
poor substrate
-
?
6-mercaptopurine ribonucleoside + H2O
6-mercaptopurine + D-ribose
show the reaction diagram
-
-
-
?
a pyrimidine nucleoside + H2O
D-ribose + a pyrimidine base
show the reaction diagram
-
-
-
-
?
adenosine + H2O
adenine + D-ribose
show the reaction diagram
adenosine + H2O
D-ribose + adenine
show the reaction diagram
-
-
-
?
cytidine + H2O
cytosine + D-ribose
show the reaction diagram
guanosine + H2O
guanine + D-ribose
show the reaction diagram
imidazoleacetic acid ribonucleotide + H2O
imidazoleacetic acid + D-ribose
show the reaction diagram
-
-
-
?
inosine + H2O
?
show the reaction diagram
catalytic efficiency towards inosine is at least 100-fold below that for uridine
-
-
?
inosine + H2O
D-ribose + hypoxanthine
show the reaction diagram
-
-
-
?
inosine + H2O
hypoxanthine + D-ribose
show the reaction diagram
isopentenyladenine riboside + H2O
?
show the reaction diagram
N-D-ribosylpyrimidine
pyrimidine + D-ribose
show the reaction diagram
N-D-ribosylpyrimidine + H2O
pyrimidine + D-ribose
show the reaction diagram
N-ribosylpurine + H2O
purine + D-ribose
show the reaction diagram
-
-
-
r
N-ribosylpyrimidine + H2O
pyrimidine + D-ribose
show the reaction diagram
preferred substrate
-
-
r
purine D-ribonucleosides + H2O
purine + D-ribose
show the reaction diagram
tubercidin + H2O
1-deazaadenine + D-ribose
show the reaction diagram
-
poor substrate
-
?
uridine + H2O
D-ribose + uracil
show the reaction diagram
-
-
-
?
uridine + H2O
uracil + D-ribose
show the reaction diagram
xanthosine + H2O
xanthine + D-ribose
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
a pyrimidine nucleoside + H2O
D-ribose + a pyrimidine base
show the reaction diagram
-
-
-
-
?
N-D-ribosylpyrimidine
pyrimidine + D-ribose
show the reaction diagram
N-ribosylpurine + H2O
purine + D-ribose
show the reaction diagram
P33022
-
-
-
r
N-ribosylpyrimidine + H2O
pyrimidine + D-ribose
show the reaction diagram
P33022
preferred substrate
-
-
r
additional information
?
-
P33022
the enzyme is required for recycling of nitrogenous bases
-
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Na+
-
likely bound to the enzyme under physiological conditions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,4-diaminophenyl-D-iminoribitol
-
competitive, the ligand can bind at the active site in two distinct orientations, binding structure, overview
4,6-Dihydroxy-1-beta-D-ribofuranosylpyrazolo-(3,4-d) pyrimidine
-
-
4-Hydroxyl-1-beta-D-ribofuranosylpyrazolo-(3,4-d) pyrimidine
-
-
4-Methylthio-1-beta-D-ribofuranosylpyrazolo-(3,4-d)pyrimidine
-
-
4-Thio-1-beta-D-ribofuranosylpyrazolo-(3,4-d)pyrimidine
-
-
adenosine
AgCl
-
at 1 mM: inhibition less than 10%
CaCl2
-
at 1 mM, inhibition less than 10%
CdCl2
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
CoCl2
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
CuCl2
-
at 1 mM: inhibition less than 10%
cytidine
-
at 6.25 mM, competitive inhibitor for uridine hydrolysis
EDTA
-
at 1 mM: inhibits uridine hydrolysis by 50%
FeCl3
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
FeSO4
-
at 1 mM: inhibition less than 10%
guanosine
-
-
HgCl2
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
Inosine
-
at 6.25 mM, competitive inhibitor for uridine hydrolysis
MgCl2
-
at 1 mM: inhibition less than 10%
MnSO4
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
NH4Cl
-
at 1 mM: inhibition less than 10%
ZnCl2
-
at 1 mM: inhibition ranging from 40-95%, at 0.01 mM: inhibition less than 10%
additional information
-
poor inhibitors
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.5
1-beta-D-ribofuranosylthymine
-
-
0.18
4-nitrophenyl ribopyranoside
pH 7.0, 35C, recombinant enzyme
0.186 - 2.5
5-Bromouridine
0.128
5-fluorouridine
-
pH 7.3, 37C
0.22
5-iodouridine
-
pH 7.3, 37C
0.329
5-methyluridine
-
pH 7.3, 37C
0.33
6-mercaptopurine ribonucleoside
-
-
0.5 - 0.7
adenosine
0.524 - 1
cytidine
0.363 - 0.6
guanosine
0.2 - 5.3
Inosine
0.4
isopentenyladenine-riboside
recombinant enzyme expressed in Escherichia coli, in 50 mM Tris-HCl, pH 7.5, at 30C
0.44
methyluridine
-
isoform NSH1, pH not specified in the publication, at 30C
0.78
purine ribonucleoside
-
-
0.083 - 5.5
uridine
1.11 - 6.5
Xanthosine
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
39
4-nitrophenyl ribopyranoside
Escherichia coli K-12
P33022
pH 7.0, 35C, recombinant enzyme
30.8
5-Bromouridine
Escherichia coli
-
pH 7.3, 37C
14.7
5-fluorouridine
Escherichia coli
-
pH 7.3, 37C
42.7
5-iodouridine
Escherichia coli
-
pH 7.3, 37C
25.5
5-methyluridine
Escherichia coli
-
pH 7.3, 37C
0.0154
adenosine
Escherichia coli K-12
P33022
pH 7.0, 35C, recombinant enzyme
11.6 - 39.4
cytidine
0.0056
guanosine
Escherichia coli K-12
P33022
pH 7.0, 35C, recombinant enzyme
0.035 - 3.62
Inosine
4.4 - 72.9
uridine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.085
3,4-diaminophenyl-D-iminoribitol
-
pH 7.4, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
-
substrate: uridine
14.5
purified recombinant enzyme, substrate inosine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
-
substrate: inosine
6.2
-
substrate: cytidine and uridine
7.4
-
assay at
8
-
substrate: 1-beta-D-ribofuranosylthymine
8.5
-
substrate: cytidine, uridine, 5-bromouridine and adenosine
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
40
-
substrate: cytidine and uridine
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 110
-
60C: about 50% of maximal activity, 110C: about 60% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2
-
calculated from sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
URH1 is mainly transcribed in the vascular cells of roots
Manually annotated by BRENDA team
of roots and in root tips
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
isoform NSH2
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35210
-
4 * 35210, calculated from sequence
62000
-
gel filtraton
137000
-
calculated from amino acid sequence
140000
-
gel filtration
143000
180000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
-
hanging drop vapour diffusion method, using 100 mM Tris (pH 8.5), 200 mM NaCl, and 24% PEG 4000
RihA bound to inhibitor 3,4-diaminophenyl-D-iminoribitol, hanging drop vapour diffusion method, 8 mg/ml RihA in 50 mM HEPES, pH 7.2, 150 mM NaCl is mixed with a 5:1 molar excess of 3,4-diaminophenyl-D-iminoribitol, solubilized in 50 mM HEPES, pH 7.2, and incubated at 4C for 3 hours, the protein/inhibitor complex is mixed with an equal volume of a precipitant solution containing 25% PEG 4000, 0.1 M sodium acetate, pH 5.0, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement
-
purified detagged recombinant enzyme, hanging drop vapour diffusion method, 10 mg/ml protein in 20 mM HEPES, pH 7.4, 150 mM NaCl, is mixed with precipitant solution, 25C, equilibration versus reservoir solution containing 100 mM Tris-HCl, pH 8.5, 200 mM NaCl, 25% w/v PEG 3350, cryoprotection by 25% glycerol, X-ray diffraction structure determination and analysis at 1.7 A resolution
-
purified recombinant enzyme in complex with D-ribose, hanging drop vapour diffusion method, 10 mg/ml protein in 10 mM Tris, pH 7.0, 25 mM NaCl, and 500 mM D-ribose, is mixed with an equal volume of precipitant solution containing 24% 2-methyl-2,4-pentanediol, 0.1 M sodium acetate, pH 5.0, and 500 mM D-ribose, 20C, 1 week, X-ray diffraction structure determination and analysis at 1.78 A resolution
crystal structure is determined at 1.6 A resolution. The enzyme is crystallized using the hanging drop vapor diffusion method by mixing an equal amount of protein and a precipitant solution, composed of 100 mM bicine (pH 9) and 1.5 M ammonium sulfate
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
-
for 10 min at pH 5.0, 50% of activity remains; for 10 min at pH 7.0-9.2, more than 90% of activity remains
50
-
for 90 min, 10% of activity remains
55
-
after 2 min: all of the uridine-cleaving activity and 62% of the inosine-cleaving activity remains
60
-
for 15 min, 60% denaturation
70
-
for 5 min at pH 8.0, complete inactivation
90
-
1 h, stable
100
-
half-life: 37 min
108
-
no thermal unfolding transition can be obtained up to 108C
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
guanidine-HCl
-
guanidine hydrochloride-induced unfolding is reversible
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, 1-3 mg of protein per ml, stable for 6 months
-
3C, cell extract heated for 10 min at 55C before passage through a DEAE-cellulose column, enzyme thus purified loses 5% of activity after 30 days
-
4C, 0.032 mg protein per ml, over 70% of activity remained after 1 month
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography
Ni-NTA column chromatography and MonoQ column chromatography
partial purification, 300fold
-
recombinant enzyme
-
recombinant enzyme from strain W6 by nickel chelate affinity chromatography and gel filtration
recombinant His6-tagged enzyme from strain BL21(DE3) by nickel chelate affinity chromatography and gel filtration, the His-tag is cleaved off by thrombin
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned and expressed in Escherichia coli
-
expressed in Escherichia coli and Saccharomyces cerevisiae; URH1, DNA and amino acid sequence determination and analysis, expression in Escherichia coli, functional complementation of a yeast mutant, expression in transgenic Arabidopsis thaliana mutant plants under control of the CaMV 35S promoter
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
gene ybeK, expression in strain W6
gene yeiK, subcloning in strain DH5alpha, expression of the His6-tagged enzyme in strain BL21(DE3)
-
overexpressed in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H239A
-
dramatic increase in Km for uridine, unchanged kcat
H82N
-
small increase in Km, increase in kcat
Q227A
the mutation causes an increase of kcat for uridine and inosine
Q227F
the mutation causes an increase of kcat for uridine and inosine
Q227Y
the mutation has a strong, enhancing effect on the hydrolysis of inosine, and the catalytic efficiency for the purinic substrate is increased by a factor of 7.6
T223A
the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine
T223F
the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine
T223F/Q227Y
the mutant shows a 2fold increase in catalytic efficiency toward inosine
T223Y
the mutation does not affect the specificity of the enzyme toward inosine or uridine
T223Y/Q227Y
the mutant displays a catalytic efficiency toward inosine that is more than 50fold increased compared to that of wild type enzyme
T227A
the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine
T227F
the mutation does not improve significantly the catalytic efficiency of YeiK toward inosine
additional information
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