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Information on EC 2.7.4.22 - UMP kinase and Organism(s) Escherichia coli and UniProt Accession P0A7E9
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2.7.4.22 UMP kinaseIUBMB Comments
This enzyme is strictly specific for UMP as substrate and is used by prokaryotes in the de novo synthesis of pyrimidines, in contrast to eukaryotes, which use the dual-specificity enzyme UMP/CMP kinase (EC 2.7.4.14) for the same purpose . This enzyme is the subject of feedback regulation, being inhibited by UTP and activated by GTP .
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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
umpk, ump kinase, uridine monophosphate kinase, uridylate kinase, ump-kinase, umpks, ssumpk, xc1936, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
ATP:UMP phosphotransferase
This enzyme is strictly specific for UMP as substrate and is used by prokaryotes in the de novo synthesis of pyrimidines, in contrast to eukaryotes, which use the dual-specificity enzyme UMP/CMP kinase (EC 2.7.4.14) for the same purpose [2]. This enzyme is the subject of feedback regulation, being inhibited by UTP and activated by GTP [1].
CAS REGISTRY NUMBER
COMMENTARY
9036-23-1
-
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
ATP + UMP
ADP + UDP
at 30°C, pH 7.4, in the presence of 2 mM MgCl2
-
-
?
ATP + UMP
ADP + UDP
the enzyme regulatory mechanisms involve GTP and UTP, overview
-
-
?
ATP + UMP
ADP + UDP
movements induced by binding uridine nucleotides, GTP and UTP interaction mechanism, analysis, overview
-
-
?
additional information
?
-
no reaction with dUMP, CMP, dCMP or TMP as substrate
-
-
?
additional information
?
-
-
no reaction with dUMP, CMP, dCMP or TMP as substrate
-
-
?
additional information
?
-
Asp93 appears as one of the key amino acids in the transfer of information from the regulatory site to the active site
-
-
?
additional information
?
-
-
Asp93 appears as one of the key amino acids in the transfer of information from the regulatory site to the active site
-
-
?
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
ATP + UMP
ADP + UDP
the enzyme regulatory mechanisms involve GTP and UTP, overview
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
UMP
excess UMP at high concentrations, UTP deminishes this inhibition
UTP
0.25 mM, 0.5 mM, GTP, UMP and high concentrations of Mg2+ protects against inhibition by UTP, decreases the affinity for ATP, inhibition only without Mg2+
UTP
the inhibitory effect is maximal at pH 8 and a low (0.1 mM) UMP concentration
UTP
Mg-free competitive cooperative inhibitor, the inhibition is reversible by GTP. UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
dialdehyde-GTP
slight stimulatory effect, 30% maximal increase in UMP-kinase activity
GTP
0.5 mM, reverses the inhibition of excess UMP, it increases the affinity for ATP
GTP
allosteric activator, the GTP-binding site, situated at 15 A from the UMP-binding site and at 24 A from the ATP-binding site, is delineated by two contiguous dimers. movements induced by binding GTP, overview. UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.23
MgATP2-
N140A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
0.26
MgATP2-
D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
0.59
MgATP2-
D93A/D159N mutant protein, 0.3 mM UMP, 0.5 mM GTP, 0.5 mM UTP
0.0755
UMP
D93A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP
0.1086
UMP
N140A/D159N mutant protein, 0.2 mM ATP, 0.5 mM GTP, 0.5 mM UTP
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100.3
105
128
153
61.6
8.2
84.2
92.5
additional information
additional information
R92A, H96A, and R127A exhibit increased enzyme activity
additional information
-
R92A, H96A, and R127A exhibit increased enzyme activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
150000
25800
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexamer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
bound to the UMP substrate, resolved at 2.3 A resolution, bound to the UDP product, resolved at 2.6 A resolution, bound to UTP, resolved at 2.45 A resolution
purified recombinant UMP kinase mutant D159N, bound to GTP, by hanging drop vapour diffusion method, 0.004 ml of protein solution containing 4 mg/ml UMPK mutant in 50 mM Tris-HCl, pH 8.0, and 100 mM NaCl, mixed with 0.004 ml precipitant solution containing 22.5 mM GTP and 21.5% w/v PEG 400 in 100 mM sodium acetate, pH 4.6, equilibration against reservoir solution containing 43% PEG 400, 20°C, X-ray diffraction structure determination and analysis at 2.8-3.0 A resolution, molecular replacement and structure modelling
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D115A
little detrimental effect, activated by 5'-guanylyl-imidodiphosphate within a concentration range that is roughly similar to that of wild type enzyme
D146N
84% of wild-type activity in the pellet of the sonicated bacterial extract
D159N
D168N
D174N
D201N
D77N
D93A
site-directed mutagenesis, inhibition by UTP appears significantly altered in the case of the D93A mutant as compared to the wild-type enzyme, the D93A substitution completely suppresses the related subunit-subunit hydrogen bonds between its main chain and Asp93, no inhibition by UTP in presence or absence of GTP. The Tm of the D93A variant is 10°C lower than that of the wild-type enzyme
D93A/D159N
D93 involved in hydrogen bond between the subunits of a dimer, mutation decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition
G232D
resistance to heat denaturation is altered, catalytic avtivity is reduced to 17% of the wild-type
L226Q
more insoluble than wild-type, impairs the stability of the enzyme
N111A
little detrimental effect, activated by 5'-guanylyl-imidodiphosphate within a concentration range that is roughly similar to that of wild type enzyme
N140A
N140A/D159N
in N140A mutant protein is the cooperativity of inhibition caused by UTP suppressed
N72A
site-directed mutagenesis, no inhibition by UTP in presence or absence of GTP
N72A/D93A
site-directed mutagenesis, the N72A mutation has less severe effects on enzyme activity regulation than the D93A substitution, reduced inhibition by UTP in absence of GTP, no inhibition in presence of GTP. The Tm of the mutant variant is 15°C lower than that of the wild-type enzyme
N72A/D93A/D159N
N72, D93 involved in hydrogen bonds between the subunits
P141L
affects enzyme activity and especially the allosteric regulation
R11H
lowered catalytic activity, 45% of the wild-type, resistance to heat denaturation is impaired
R62H
T138A
decreases half-denaturation temperature of UMP kinase by around 10°C, results in 4times higher Km for UMP, moderate loss of sensitivity to UTP inhibition, important loss in activation by GTP
T138A/N140A
decreases half-denaturation temperature of UMP kinase by around 25°C, increases the apparant Km for ATP and UMP by a factor of 2.6 and 12, respectively
N140A
cooperative inhibition by GTP and UTP is altered, lower thermodynamic stability
D159N
exhibits higher solubility than the wild-type protein at neutral pH, 10% of wild-type activity in the pellet of the sonicated bacterial extract
D168N
97% of wild-type activity in the pellet of the sonicated bacterial extract
D174N
loss of two-thirds of its activity after 3 months storage at 4°C in 50 mM Tris-HCl (pH 7.4), 98% of wild-type activity in the pellet of the sonicated bacterial extract
D201N
exhibits 10% of the wild-type activity, has altered stability and regulatory properties
D201N
exhibits higher solubility than the wild-type protein at neutral pH, 42% of wild-type activity in the pellet of the sonicated bacterial extract
D77N
84% of wild-type activity in the pellet of the sonicated bacterial extract, insensitive to activation by GTP
D77N
affects enzyme activity and especially the allosteric regulation
N140A
decreases half-denaturation temperature of UMP kinase by around 10°C, moderate loss of sensitivity to UTP inhibition, important loss in activation by GTP
N140A
site-directed mutagenesis, UTP and GTP are tightly coupled in both wild-type enzyme and N140A variant. The Tm of the mutant variant is 10°C lower than that of the wild-type enzyme
R62H
83% of wild-type activity in the pellet of the sonicated bacterial extract, insensitive to activation by GTP
R62H
as stable as wild-type, affects enzyme activity and especially the allosteric regulation
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
59 - 62
mutants R62H, D146N, D168N and D174N are half inactivated between 59 and 62°C
63
additional information
additional information
thermal stability of the N72A/D159N variant is close to that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 10°C lower than that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 15°C lower than that of the D159N variant,
additional information
-
thermal stability of the N72A/D159N variant is close to that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 10°C lower than that of the D159N variant, temperature of half-inactivation of the D93A/D159N variant is 15°C lower than that of the D159N variant,
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 50 mM Tris-HCl (pH 7.4) as insoluble proteins, after solubilization with 0.1 M borate (pH 9) or with 1 mM UTP in 50 mM Tris-HCl (pH 7.4), the enzyme is fully active
4°C, 50 mM Tris-HCl (pH 7.4), several months, no significant loss of activity. An exception is mutant D174N, which loses two-thirds of its activity after 3 months at 4°C
room temperature, 0.1 M borate buffer (pH 8.5), 2 months, no loss of activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
of the recombinant protein by affinity chromatography, no difference in activity compared to the wild type
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
His-tagged version expressed in Escherichia coli BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
drug development
as bacterial UMP kinases have no counterpart in eukaryotes, the information provided here can help the design of new antibiotics
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Labesse, G.; Bucurenci, N.; Douguet, D.; Sakamoto, H.; Landais, S.; Gagyi, C.; Gilles, A.M.; Barzu, O.
Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase
Biochem. Biophys. Res. Commun.
294
173-179
2002
Escherichia coli (P0A7E9), Escherichia coli
Serina, L.; Blondin, C.; Krin, E.; Sismeiro, O.; Danchin, A.; Sakamoto, H.; Gilles, A.M.; Barzu, O.
Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP
Biochemistry
34
5066-5074
1995
Escherichia coli (P0A7E9), Escherichia coli
Serina, L.; Bucurenci, N.; Gilles, A.M.; Surewicz, W.K.; Fabian, H.; Mantsch, H.H.; Takahashi, M.; Petrescu, I.; Batelier, G.; Barzu, O.
Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP
Biochemistry
35
7003-7011
1996
Escherichia coli (P0A7E9), Escherichia coli
Gagyi, C.; Bucurenci, N.; Sirbu, O.; Labesse, G.; Ionescu, M.; Ofiteru, A.; Assairi, L.; Landais, S.; Danchin, A.; Barzu, O.; Gilles, A.M.
UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity
Eur. J. Biochem.
270
3196-3204
2003
Bacillus subtilis (O31749), Bacillus subtilis, Escherichia coli (P0A7E9), Escherichia coli
Bucurenci, N.; Serina, L.; Zaharia, C.; Landais, S.; Danchin, A.; Barzu, O.
Mutational analysis of UMP kinase from Escherichia coli
J. Bacteriol.
180
473-477
1998
Escherichia coli (P0A7E9), Escherichia coli
Landais, S.; Gounon, P.; Laurent-Winter, C.; Mazie, J.C.; Danchin, A.; Barzu, O.; Sakamoto, H.
Immunochemical analysis of UMP kinase from Escherichia coli
J. Bacteriol.
181
833-840
1999
Escherichia coli (P0A7E9), Escherichia coli
Briozzo, P.; Evrin, C.; Meyer, P.; Assairi, L.; Joly, N.; Barzu, O.; Gilles, A.M.
Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation
J. Biol. Chem.
280
25533-25540
2005
Escherichia coli (P0A7E9), Escherichia coli
Sakamoto, H.; Landais, S.; Evrin, C.; Laurent-Winter, C.; Barzu, O.; Kelln, R.A.
Structure-function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria
Microbiology
150
2153-2159
2004
Escherichia coli (P0A7E9), Salmonella enterica subsp. enterica serovar Typhimurium (P65933)
Evrin, C.; Straut, M.; Slavova-Azmanova, N.; Bucurenci, N.; Onu, A.; Assairi, L.; Ionescu, M.; Palibroda, N.; Barzu, O.; Gilles, A.
Regulatory mechanisms differ in UMP kinases from Gram-negative and Gram-positive bacteria
J. Biol. Chem.
282
7242-7253
2007
Streptococcus pneumoniae, Enterococcus faecalis, Haemophilus influenzae, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium, Escherichia coli (A7ZWB7), Bacillus subtilis (O31749), Neisseria meningitidis (P65932)
Meyer, P.; Evrin, C.; Briozzo, P.; Joly, N.; Barzu, O.; Gilles, A.M.
Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP
J. Biol. Chem.
283
36011-36018
2008
Escherichia coli (P0A7E9), Escherichia coli
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