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Information on EC 3.2.2.8 - ribosylpyrimidine nucleosidase and Organism(s) Escherichia coli and UniProt Accession P33022
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3.2.2.8 ribosylpyrimidine nucleosidaseIUBMB Comments
Also hydrolyses purine D-ribonucleosides, but much more slowly. 2'-, 3'- and 5'-deoxynucleosides are not substrates .
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Word Map
- 3.2.2.8
- purine
- leishmania
-
salvage
- inosine
- leishmaniasis
- donovani
- crithidia
- fasciculata
- vivax
-
purine-specific
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n-glycosidic
- ribonucleosides
-
schramm
-
chaga
-
horenstein
- iminoribitols
-
inosine-adenosine-guanosine
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ribooxocarbenium
- xanthosine
-
zymodeme
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
nucleoside hydrolase, cu-nh, n-ribohydrolase, sscu-nh, pyrimidine-specific nucleoside hydrolase, pyrimidine nucleoside hydrolase, sso0505, uridine-ribohydrolase 1, cytidine-uridine nucleoside hydrolase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
N-ribosylpyrimidine nucleosidase
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N-ribosylpyrimidine ribohydrolase
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nucleosidase, pyrimidine
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pyrimidine nucleosidase
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YeiK
additional information
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the enzyme belongs to the pyrimidine-preferring N-ribohydrolases, CU-NHs, a class of Ca2+-dependent enzymes that catalyze the hydrolytic cleavage of the N-glycosidic bond in pyrimidine nucleosides
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of N-glycosyl bond
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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
37288-60-1
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
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enzyme is pyrimidine specific, purine nucleosides are not hydrolysed, 2'-, 3'- and 5'-deoxynucleosides are no substrates
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?
additional information
?
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catalytic cycles between the open and closed conformations of RihA, stabilization of two flexible active site regions is pivotal to establish the interactions required for substrate discrimination and catalysis, involvement of the Asp10 as general base in the mechanism, role of the conserved His82 residue in modulating product release, structure-function analysis, detailed overview
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
the substrate binds to the Ca2+-containing active site in the catalytic cavity at the C-terminal end of the core beta-sheet
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3,4-diaminophenyl-D-iminoribitol
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competitive, the ligand can bind at the active site in two distinct orientations, binding structure, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.16
Inosine
mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.77
Inosine
mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.93
Inosine
mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
2.14
Inosine
mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
2.34
Inosine
wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C
3.29
Inosine
mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C
4.31
Inosine
mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C
4.42
Inosine
mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
5.3
Inosine
mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.12
uridine
wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.31
uridine
mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.33
uridine
mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.57
uridine
mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.77
uridine
mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.06
uridine
mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.09
uridine
mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.13
uridine
mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
1.19
uridine
mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.035
Inosine
mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.086
Inosine
wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.109
Inosine
mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.125
Inosine
mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.18
Inosine
mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.182
Inosine
mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.382
Inosine
mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
0.593
Inosine
mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
3.62
Inosine
mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
5.4
uridine
wild type enzyme, in 50 mM HEPES buffer (pH 7.3), at 37°C
15.1
uridine
mutant enzyme Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
18.8
uridine
mutant enzyme T223F, in 50 mM HEPES buffer (pH 7.3), at 37°C
39.8
uridine
mutant enzyme T223A, in 50 mM HEPES buffer (pH 7.3), at 37°C
44.3
uridine
mutant enzyme T223Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
46.9
uridine
mutant enzyme Q227A, in 50 mM HEPES buffer (pH 7.3), at 37°C
52.5
uridine
mutant enzyme Q227F, in 50 mM HEPES buffer (pH 7.3), at 37°C
59.1
uridine
mutant enzyme T223F/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
72.9
uridine
mutant enzyme T223Y/Q227Y, in 50 mM HEPES buffer (pH 7.3), at 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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possible role of CU-NHs in the breakdown of modified nucleosides derived from RNA molecules
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
tetramer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method, using 100 mM Tris (pH 8.5), 200 mM NaCl, and 24% PEG 4000
molecular dynamics simulation. Both in wild-type and mutant T223Y/Q227Y, inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and pi-interactions between residues His82 and His239 and the nucleobase. The lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. A hydrogen-bonding network between hypoxanthine and a general acid Asp15 is present when the two Tyr mutations are engineered into the active site. This hydrogen-bonding network is only maintained when both Tyr mutations are present due to a pi-interaction between the residues
QM/MM simulations. The relatively stronger hydrogen-bond interactions between uridine and the active-site residues Gln227 and Tyr231 play an important role in enhancing the substrate binding and thus promoting the N-glycosidic bond cleavage, in comparison with inosine. The estimated energy barrier is 30 kcal/mol for the hydrolysis of inosine and 22 kcal/mol for uridine. The uridine binding is exothermic by about 23 kcal/mol, and inosine binding by 12 kcal/mol
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 4°C 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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
T223Y/Q227Y
the mutant displays a catalytic efficiency toward inosine that is more than 50fold increased compared to that of wild type enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Giabbai, B.; Degano, M.
Cloning, purification, crystallization and x-ray analysis of the Escherichia coli pyrimidine nucleoside hydrolase YeiK
Acta Crystallogr. Sect. D
D60
524-527
2004
Escherichia coli
Giabbai, B.; Degano, M.
Crystal structure to 1.7 ANG of the Escherichia coli pyrimidine nucleoside hydrolase YeiK, a novel candidate for cancer gene therapy
Structure
12
739-749
2004
Escherichia coli
Iovane, E.; Giabbai, B.; Muzzolini, L.; Matafora, V.; Fornili, A.; Minici, C.; Giannese, F.; Degano, M.
Structural basis for substrate specificity in group I nucleoside hydrolases
Biochemistry
47
4418-4426
2008
Escherichia coli (P33022), Escherichia coli
Garau, G.; Muzzolini, L.; Tornaghi, P.; Degano, M.
Active site plasticity revealed from the structure of the enterobacterial N-ribohydrolase RihA bound to a competitive inhibitor
BMC Struct. Biol.
10
14
2010
Escherichia coli
Lenz, S.A.P.; Wetmore, S.D.
Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase insights from molecular dynamics simulations
J. Comput. Aided Mol. Des.
32
1375-1388
2018
Escherichia coli (P33022), Escherichia coli
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