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Information on EC 2.4.2.3 - uridine phosphorylase and Organism(s) Escherichia coli and UniProt Accession P12758

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     2 Transferases
         2.4 Glycosyltransferases
             2.4.2 Pentosyltransferases
                2.4.2.3 uridine phosphorylase
IUBMB Comments
The enzyme participates the the pathways of pyrimidine ribonucleosides degradation and salvage. The mammalian enzyme also accepts 2'-deoxyuridine.
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This record set is specific for:
Escherichia coli
UNIPROT: P12758
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Synonyms
uridine phosphorylase, uph, upase, urdpase, uridine phosphorylase 1, pcup1, stuph, l-urdpase, vchuph, upase-2, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphorylase, uridine
-
-
-
-
pyrimidine phosphorylase
-
-
-
-
UDRPase
-
-
-
-
UPase
UPH
-
-
-
-
UrdPase
-
-
-
-
uridine:orthophosphate alpha-D-ribosyltransferase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
uridine + phosphate = uracil + alpha-D-ribose 1-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pentosyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -, -
SYSTEMATIC NAME
IUBMB Comments
uridine:phosphate alpha-D-ribosyltransferase
The enzyme participates the the pathways of pyrimidine ribonucleosides degradation and salvage. The mammalian enzyme also accepts 2'-deoxyuridine.
CAS REGISTRY NUMBER
COMMENTARY hide
9030-22-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
uridine + phosphate
uracil + alpha-D-ribose 1-phosphate
show the reaction diagram
2'-deoxyuridine + phosphate
uracil + deoxyribose 1-phosphate
show the reaction diagram
-
6% of the activity with uridine
-
?
5-bromo-2'-deoxyuridine + phosphate
5-bromouracil + 2-deoxribose 1-phosphate
show the reaction diagram
-
27% of the activity with uridine
-
-
?
5-bromouridine + phosphate
5-bromouracil + alpha-D-ribose 1-phosphate
show the reaction diagram
-
69% of the activity with uridine
-
-
?
5-fluoro-2'-deoxyuridine + phosphate
5-fluorouracil + 2-deoxyribose-1-phosphate
show the reaction diagram
-
14% of the activity with uridine
-
-
?
5-methyluridine + phosphate
thymine + alpha-D-ribose 1-phosphate
show the reaction diagram
-
19% of the activity with uridine
-
-
?
thymidine + phosphate
thymine + 2-deoxy-alpha-D-ribose 1-phosphate
show the reaction diagram
-
2% of the activity with uridine
-
-
?
uridine + phosphate
uracil + alpha-D-ribose 1-phosphate
show the reaction diagram
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
uridine + phosphate
uracil + alpha-D-ribose 1-phosphate
show the reaction diagram
key enzyme in pyrimidine-salvage pathway
-
-
?
uridine + phosphate
uracil + alpha-D-ribose 1-phosphate
show the reaction diagram
-
-
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-benzylacyclouridine
-
5-m-benzyloxybenzyl acyclouridine
-
5-m-benzyloxybenzyl barbituric acid acyclonucleoside
-
5-phenylselenenylacyclouridine
-
5-phenylthioacyclouridine
-
1-(1',2'-dihydroxypropyl)-5,6-tetramethyleneuracil
-
only the R-enantiomer inhibits, but not the S-enantiomer
1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil
-
inhibits forward and reverse reaction
Acyclonucleoside analogues
-
consisting of 5- and 5,6-substituted uracils and different acyclic chains
-
guanidine hydrochloride
-
50% loss of activity at 1.04 M, only little residual activity between 2 and 6 M
N-ethyl-5-phenylisoxazolium-3'-sulfonate
-
Woodward's reagent K
phosphate
-
product inhibition
ribose 1-phosphate
-
product inhibition
Uracil
-
product inhibition
uridine
-
product inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.9
phosphate
-
-
0.91
uridine
-
-
additional information
additional information
-
-
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0043
5-benzylacyclouridine
-
0.00068
5-m-benzyloxybenzyl acyclouridine
-
0.0027
1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil
-
-
0.07 - 2.5
N-ethyl-5-phenylisoxazolium-3'-sulfonate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100 - 120
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
-
uridine phosphorolysis
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.6 - 8.4
-
mutant UPL8, more than 60% of maximum activity within
5.8 - 9
-
pH 5.8: about 50% of activity maximum, pH 9.0: about 40% of activity maximum
6 - 8.2
-
wild-type, more than 60% of maximum activity within
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
40
-
wild-type
60
-
mutant UPL8
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
130000
-
gel filtration, native enzyme
160000
-
gel filtration
22200
-
8 * 22200, SDS-PAGE
27000
-
x * 27000, SDS-PAGE
27500
-
6 * 27500
29000
-
x * 29000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
monomer
-
in the presence of 2 M guanidine hydrochloride
octamer
-
8 * 22200, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method, crystals of thymidine phosphate complex, 2'-deoxyuridine complex, 5-fluorouracil ribose 1-phosphate complex and native enzyme form
hanging-drop vapour-diffusion method, structure at 2.0 A resolution
hanging-drop vapour-diffusion method, structures of 5-benzylacyclouridine, 5-phenylthioacyclouridine, 5-phenylselenylacyclouridine, 5-m-benzyloxybenzyl acyclouridine and 5-m-benzyloxybenzyl barbituric acid acyclonucleoside bound to the active site of the enzyme, resolutions ranging from 1.95 to 2.3 A
hanging drop vapor diffusion method
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E196A
complete loss of uridine phosphorylase activity
E198D
complete loss of uridine phosphorylase activity
E198G
complete loss of uridine phosphorylase activity
E198Q
complete loss of uridine phosphorylase activity
F162A
mutation causes a drastic decrease in uridine phosphorylase activity
H8A
mutation lowers the activity by 20%
I69A
mutation does not decrease activity
M197A
low activity conserved
M197S
low activity conserved
R30A
complete loss of uridine phosphorylase activity
R30K
very low activity
R91A
complete loss of uridine phosphorylase activity
R91K
very low activity
T94A
mutation causes a drastic decrease in uridine phosphorylase activity
W196D
mutant enzyme is still partially active
Y195A
increase in activity
Y195G
mutation causes a drastic decrease in uridine phosphorylase activity
C206_A207insGVPLC
-
inactive and insoluble mutant protein
E79-E80insGVPLE
-
inactive mutant protein
G174_R175insRGTPG
-
134% of the activity of the wild-type enzyme
I102_N103insGVPHI
-
inactive and insoluble mutant protein
I98_Q99insRGTPI
-
inactive and insoluble mutant protein
K181_G182insGGTPK
-
67% of the activity of the wild-type enzyme
L9_G10insGVPHL
-
139% of the activity of the wild-type enzyme
S159_D160insGGTPS
-
inactive mutant protein
S183_M184insMGYPS
-
54% of the activity of the wild-type enzyme
S183_M184insRGTPS
-
117% of the activity of the wild-type enzyme
T12_K13insGVPLT
-
132% of the activity of the wild-type enzyme
T52_W53insWGTPT
-
inactive mutant protein
T67_G68insGGTPT
-
inactive mutant protein
V192_M193insMGYPV
-
inactive mutant protein
V31_E32insGVPRV
-
inactive and insoluble mutant protein
V42_K43insKGYPV
-
as active as the wild-type enzyme
additional information
-
evolvement of a mutant enzyme UPL8 by iterative saturation mutagenesis. Compared to the wild type enzyme, which has a temperature optimum of 40°C and a half-life of 9.89 h at 60°C, the selected mutant has a temperature optimum of 60°C and a half-life of 17.3 h at 60°C. Self-immobilization of the native enzyme as a Spherezyme shows a 3.3fold increase in thermostability while immobilized mutant enzyme shows a 4.4fold increase in thermostability
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
-
aglycone substrates, nucleoside substrates, phosphate or pentose 1-phosphate ester substrates stabilize against heat inactivation
75
-
1 h, 3.7% residual activity for wild-type, 80.2% residual activity for mutant UPL8
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
aglycone substrates, nucleoside substrates, phosphate or pentose 1-phosphate ester substrates stabilize against heat inactivation
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-73°C, stable for at least 3 months, purified enzyme
-
-73°C, stable for at least 6 months, crude extract
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
homogeneity
-
near homogeneity
-
phosphorylase type I
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
co-expressed with thymidine phosphorylase in Escherichia coli BL21(DE3) cells
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
dilution of the enzyme preincubated with 2 M guanidine hydrochloride results in partial recovery
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
-
evolvement of a mutant enzyme by iterative saturation mutagenesis. Compared to the wild type enzyme, which has a temperature optimum of 40°C and a half-life of 9.89 h at 60°C, the selected mutant has a temperature optimum of 60°C and a half-life of 17.3 h at 60°C. Self-immobilization of the native enzyme as a Spherezyme shows a 3.3fold increase in thermostability while immobilized mutant enzyme shows a 4.4fold increase in thermostability. Combining the enzyme with the purine nucleoside phosphorylase from Bacillus halodurans allows for synthesis of 5-methyluridine (a pharmaceutical intermediate) from guanosine and thymine in a one-pot transglycosylation reaction. Replacing the wild type uridine phosphorylase with the mutant allows for an increase in reaction temperature to 65°C and increased the reaction productivity from 10 to 31 g per l and h
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Krenitsky, T.A.; Tuttle, J.V.
Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases
Biochim. Biophys. Acta
703
247-249
1982
Escherichia coli
Manually annotated by BRENDA team
Mikhailov, A.M.; Smirnova, E.A.; Tsuprun, V.L.; Tagunova, I.V.; Vainshtein, B.K.; Linkova, E.V.; Komissarov, A.A.; Siprashvili, Z.Z.; Mironov, A.S.
Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12
Biochem. Int.
26
607-615
1992
Escherichia coli
Manually annotated by BRENDA team
Vita, A.; Magni, G.
A one-step procedure for the purification of uridine phosphorylase from Escherichia coli
Anal. Biochem.
133
153-156
1983
Escherichia coli, Escherichia coli B / ATCC 11303
Manually annotated by BRENDA team
Vita, A.; Huang, C.Y.; Magni, G.
Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis
Arch. Biochem. Biophys.
226
687-692
1983
Escherichia coli, Escherichia coli B / ATCC 11303
Manually annotated by BRENDA team
Cook, W.J.; Koszalka, G.W.; Hall, W.W.; Narayana, S.V.L.; Ealick, S.E.
Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli
J. Biol. Chem.
262
2852-2853
1987
Escherichia coli
Manually annotated by BRENDA team
Krenitsky, T.A.
Uridine phosphorylase from Escherichia coli. Kinetic properties and mechanism
Biochim. Biophys. Acta
429
352-358
1976
Escherichia coli
Manually annotated by BRENDA team
Drabikowska, A.K.; Lissowska, L.; Draminski, M.; Zgit-Wroblewska, L.; Shugar, D.
Acyclonucleoside analogues consisting of 5- and 5,6-substituted uracils and different acylic chains: inhibitory properties vs purified E. coli uridine phosphorylase
Z. Naturforsch. C
42c
288-296
1987
Escherichia coli, Escherichia coli B / ATCC 11303
-
Manually annotated by BRENDA team
Leer, J.C.; Hammer-Jespersen, K.; Schwartz, M.
Uridine phosphorylase from Escherichia coli. Physical and chemical characterization
Eur. J. Biochem.
75
217-224
1977
Escherichia coli
Manually annotated by BRENDA team
Burlakova, A.A.; Kurganov, B.I.; Chernyak, V.; Debabov, V.G.
Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme
Biochemistry
62
95-103
1997
Escherichia coli
Manually annotated by BRENDA team
Komissarov, A.A.; Romanova, D.V.; Debabov, V.G.
Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K
J. Biol. Chem.
270
10050-10055
1995
Escherichia coli
Manually annotated by BRENDA team
Burling, F.T.; Kniewel, R.; Buglino, J.A.; Chadha, T.; Beckwith, A.; Lima, C.D.
Structure of Escherichia coli uridine phosphorylase at 2.0 A
Acta Crystallogr. Sect. D
59
73-76
2003
Escherichia coli (P12758), Escherichia coli
Manually annotated by BRENDA team
Bu, W.; Settembre, E.C.; el Kouni, M.H.; Ealick, S.E.
Structural basis for inhibition of Escherichia coli uridine phosphorylase by 5-substituted acyclouridines
Acta Crystallogr. Sect. D
61
863-872
2005
Escherichia coli (P12758), Escherichia coli
Manually annotated by BRENDA team
Oliva, I.; Zuffi, G.; Orsini, G.; Tonon, G.; de Gioia, L.; Ghisotti, D.
Mutagenesis of Escherichia coli uridine phosphorylase by random pentapeptide insertions
Enzyme Microb. Technol.
35
309-314
2004
Escherichia coli
-
Manually annotated by BRENDA team
Oliva, I.; Zuffi, G.; Barile, D.; Orsini, G.; Tonon, G.; De Gioia, L.; Ghisotti, D.
Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis
J. Biochem.
135
495-499
2004
Escherichia coli (P12758), Escherichia coli
Manually annotated by BRENDA team
Caradoc-Davies, T.T.; Cutfield, S.M.; Lamont, I.L.; Cutfield, J.F.
Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium
J. Mol. Biol.
337
337-354
2004
Escherichia coli (P12758), Escherichia coli
Manually annotated by BRENDA team
Visser, D.; Hennessy, F.; Rashamuse, J.; Pletschke, B.; Brady, D.
Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization
J. Mol. Catal. B
68
279-285
2011
Escherichia coli
-
Manually annotated by BRENDA team
Xiong, S.; Wang, Y.; Wang, X.; Wang, J.; Li, J.; Zhang, G.; Zhang, R.; Xie, L.; Wang, H.
Enzymatic synthesis of 2-deoxyuridine by whole cell catalyst co-expressing uridine phosphorylase and thymidine phosphorylase through auto-induction system
J. Biosci. Bioeng.
118
723-727
2014
Escherichia coli
Manually annotated by BRENDA team