Information on EC 2.5.1.7 - UDP-N-acetylglucosamine 1-carboxyvinyltransferase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
2.5.1.7
-
RECOMMENDED NAME
GeneOntology No.
UDP-N-acetylglucosamine 1-carboxyvinyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
phosphoenolpyruvate + UDP-N-acetyl-alpha-D-glucosamine = phosphate + UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxyvinyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
-
-
Metabolic pathways
-
-
peptidoglycan biosynthesis
-
-
Peptidoglycan biosynthesis
-
-
UDP-N-acetylmuramoyl-pentapeptide biosynthesis I (meso-diaminopimelate containing)
-
-
UDP-N-acetylmuramoyl-pentapeptide biosynthesis II (lysine-containing)
-
-
SYSTEMATIC NAME
IUBMB Comments
phosphoenolpyruvate:UDP-N-acetyl-D-glucosamine 1-carboxyvinyltransferase
-
CAS REGISTRY NUMBER
COMMENTARY hide
9023-27-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
isoforms MurA1, murA2
-
-
Manually annotated by BRENDA team
strain T
-
-
Manually annotated by BRENDA team
strain T
-
-
Manually annotated by BRENDA team
gene murA; MurA; serovar L2
-
-
Manually annotated by BRENDA team
MurA
-
-
Manually annotated by BRENDA team
Nr. 30054
-
-
Manually annotated by BRENDA team
strain NRC 492
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain K-235
-
-
Manually annotated by BRENDA team
MurA
-
-
Manually annotated by BRENDA team
MurZ
-
-
Manually annotated by BRENDA team
strain ATCC VR1471
TrEMBL
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain Texas 26
-
-
Manually annotated by BRENDA team
Staphylococcus epidermidis Texas 26
strain Texas 26
-
-
Manually annotated by BRENDA team
strain ATCC 1470
TrEMBL
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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
phosphoenol-2-oxobutyrate + UDP-N-acetyl-D-glucosamine
phosphate + UDP-N-acetylglucosaminyl-enol-2-oxobutyrate
show the reaction diagram
-
-
-
?
phosphoenolpyruvate + UDP-N-acetyl-D-glucosamine
phosphate + UDP-N-acetyl-3-(1-carboxyvinyl)-D-glucosamine
show the reaction diagram
phosphoenolpyruvate + UDP-N-acetyl-D-glucosamine
phosphate + UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine
show the reaction diagram
phosphoenolpyruvate + UDP-N-acetyl-D-glucosamine
UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine + phosphate
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
phosphoenolpyruvate + UDP-N-acetyl-D-glucosamine
phosphate + UDP-N-acetyl-3-(1-carboxyvinyl)-D-glucosamine
show the reaction diagram
phosphoenolpyruvate + UDP-N-acetyl-D-glucosamine
phosphate + UDP-N-acetyl-3-O-(1-carboxyvinyl)-D-glucosamine
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
not affected by K+, Na+, Mg2+, and Mn2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(E)-3-fluorophosphoenolpyruvate
(S)-2-[2-(naphthalene-1-sulfonylamino)-5-(naphthalene-1-sulfonyloxy)-benzoylamino]-pentanedioic acid
-
competitive with UDP-N-acetylglucosamine
(S)-2-[2-(naphthalene-1-sulfonylamino)-5-(naphthalene-1-sulfonyloxy)-benzoylamino]-succinic acid
-
-
(Z)-3-fluorophosphoenolpyruvate
1-tuliposide A
1-tuliposide B
2-(4-methylpiperazin-1-yl)-3,4-dihydronaphthalen-1(2H)-one
2-oxo-1,3-benzoxathiol-5-yl (3-chlorophenyl)carbamate
2-oxo-1,3-benzoxathiol-5-yl methylcarbamate
2-oxo-1,3-benzoxathiol-5-yl pyridine-4-carboxylate
2-oxo-1,3-benzoxathiol-6-yl 4-nitrobenzenesulfonate
2-oxo-1,3-benzoxathiol-6-yl benzenesulfonate
2-oxo-1,3-benzoxathiol-6-yl methanesulfonate
2-oxo-1,3-benzoxathiol-6-yl sulfamate
2-[4-(2-hydroxyethyl)piperazin-1-yl]-3,4-dihydronaphthalen-1(2H)-one
2-[4-(2-hydroxyethyl)piperazin-1-yl]-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one
3-Bromopyruvate
-
irreversible, inhibitory effect is increased by UDP-GlcNAc
4,7-dichloro-5-hydroxy-1,3-benzoxathiol-2-one
5,5'-dithiobis(2-nitrobenzoic acid)
-
complete inhibition at 0.01 M; i.e. DTNB
5,7-dibromo-6-hydroxy-1,3-benzoxathiol-2-one
5-(prop-2-en-1-yloxy)-1,3-benzoxathiol-2-one
5-bromo-2-oxo-1,3-benzoxathiol-6-yl phenyl carbonate
5-hydroxy-1,3-benzoxathiol-2-one
5-hydroxy-7-(3-methylphenyl)-1,3-benzoxathiol-2-one
5-hydroxy-7-(4-methoxyphenyl)-1,3-benzoxathiol-2-one
5-hydroxybenzo[d][1,3]oxathiol-2-one
5-hydroxynaphtho[1,2-d][1,3]oxathiol-2-one
5-hydroxynaphtho[2,1-d][1,3]oxathiol-2-one
5-methoxy-1,3-benzoxathiole
5-methoxybenzo[d][1,3]oxathiol-2-one
6,7-dimethoxy-2-[4-(2-phenylethyl)piperazin-1-yl]-3,4-dihydronaphthalen-1(2H)-one
7-(4-fluorophenyl)-5-hydroxy-1,3-benzoxathiol-2-one
cnicin
-
sesquiterpene lactone. The enzyme catalyzes the formation of a covalent adduct between cnicin and substrate UDP-N-acetylglucosamine via an anti-Michael 1,3-addition of UDP-N-acetylglucosamine to an alpha,beta-unsaturated carbonyl function in cnicin thus forming a noncovalent suicide inhibitor
cnicine
Co2+
-
metal ions do not enhance the activity of enzymes, activity is inhibited by 10 mM
cynaropicrin
diarylmethane
ebselen
fosfomycin
HESFWYLPHHQSY
imidazole
iodoacetamide
-
inhibition by alkylation of the active site Cys155, pH-dependent, no alkylation below pH 7.0, maximum alkylation at pH 9.0
iodoacetate
-
-
N-ethylmaleimide
p-chloromercuribenzoate
-
-
PEP 1354 peptide
-
competitive inhibitor
-
PGE-553828
-
competitive against UDP-GlcNAc; inhibition mechanism, kinetics
-
phosphoenol-2-ketovalerate
-
-
-
phosphonomycin
pyrazolopyrimidine
RWJ-110192
RWJ-140998
RWJ-3981
terreic acid
thimerosal
thiram
Trypsin
-
wild-type and mutant C115S, no protection via individually binding of substrates or inhibitor fosfomycin, but via binding of both, the 2 substrates and inhibitor fosfomycin
-
tulipaline A
tulipaline B
UDP-N-acetylmuramic acid
UDP-N-acetylmuramic acid-L-Ala
-
weak
UDP-N-acetylmuramic acid-L-Ala-D-Glu
UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-alpha,epsilon-diaminopimelic acid
uridine diphospho-N-acetylmuramyl-L-Ala-D-gamma-Glu-meso-alpha,epsilon-diaminopimelic-acid-D-Ala-D-Ala
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
thiol groups
-
required for activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0004 - 1
phosphoenolpyruvate
0.0057 - 2.5
UDP-GlcNAc
0.015 - 0.244
UDP-N-acetyl-D-glucosamine
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.76 - 1.13
fosfomycin
0.41 - 4.75
phosphoenolpyruvate
0.78 - 8.9
UDP-GlcNAc
0.41 - 4.75
UDP-N-acetyl-D-glucosamine
additional information
additional information
Enterobacter cloacae
-
kcat values of mutant enzymes for the substrates
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000005 - 0.01
phosphoenolpyruvate
0.0000017 - 0.00025
UDP-N-acetyl-D-glucosamine
520000 - 6000000
additional information
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.1
(Z)-3-fluorophosphoenolpyruvate
0.0086 - 2
fosfomycin
0.038
PGE-553828
-
-
-
1.7
phosphoenol-2-ketovalerate
-
-
-
0.016
T6362
-
20C, pH 8.0
4.9 - 6.6
UDP-N-acetylmuramyl-L-Ala-D-Glu-meso-alpha,epsilon-diaminopimelic acid
-
-
0.8
uridine diphospho-N-acetylmuramyl-L-Ala-D-gamma-Glu-meso-alpha,epsilon-diaminopimelic-acid-D-Ala-D-Ala
-
-
additional information
additional information
-
-
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00853 - 0.01204
2-(4-methylpiperazin-1-yl)-3,4-dihydronaphthalen-1(2H)-one
0.00286
2-oxo-1,3-benzoxathiol-5-yl (3-chlorophenyl)carbamate
0.00046
2-oxo-1,3-benzoxathiol-5-yl methylcarbamate
0.00058
2-oxo-1,3-benzoxathiol-5-yl pyridine-4-carboxylate
0.00515 - 0.01079
2-oxo-1,3-benzoxathiol-6-yl 4-nitrobenzenesulfonate
0.00196 - 0.00668
2-oxo-1,3-benzoxathiol-6-yl benzenesulfonate
0.00076 - 0.00113
2-oxo-1,3-benzoxathiol-6-yl methanesulfonate
0.00025 - 0.01794
2-oxo-1,3-benzoxathiol-6-yl sulfamate
0.01252 - 0.02249
2-[4-(2-hydroxyethyl)piperazin-1-yl]-3,4-dihydronaphthalen-1(2H)-one
0.00313 - 0.01735
2-[4-(2-hydroxyethyl)piperazin-1-yl]-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one
0.00028 - 0.00109
4,7-dichloro-5-hydroxy-1,3-benzoxathiol-2-one
0.00095 - 0.00349
5,7-dibromo-6-hydroxy-1,3-benzoxathiol-2-one
0.00801
5-(prop-2-en-1-yloxy)-1,3-benzoxathiol-2-one
0.00727
5-hydroxy-7-(3-methylphenyl)-1,3-benzoxathiol-2-one
Escherichia coli
-
in 50 mM Tris-HCl, pH 7.9, at 37C
0.00951
5-hydroxy-7-(4-methoxyphenyl)-1,3-benzoxathiol-2-one
Escherichia coli
-
in 50 mM Tris-HCl, pH 7.9, at 37C
0.00053 - 0.02707
5-hydroxybenzo[d][1,3]oxathiol-2-one
0.0031 - 0.02431
5-hydroxynaphtho[1,2-d][1,3]oxathiol-2-one
0.00155 - 0.02609
5-hydroxynaphtho[2,1-d][1,3]oxathiol-2-one
0.01428
5-methoxy-1,3-benzoxathiole
Escherichia coli
-
in 50 mM Tris-HCl, pH 7.9, at 37C
0.00645 - 0.02479
5-methoxybenzo[d][1,3]oxathiol-2-one
0.02256 - 0.029
6,7-dimethoxy-2-[4-(2-phenylethyl)piperazin-1-yl]-3,4-dihydronaphthalen-1(2H)-one
0.01854
7-(4-fluorophenyl)-5-hydroxy-1,3-benzoxathiol-2-one
Escherichia coli
-
in 50 mM Tris-HCl, pH 7.9, at 37C
0.0001
ebselen
Haemophilus influenzae
-
50 mM Tris-HCl, pH 7.8, compound displays an anti-MurA activity that is above 90% at a concentration below 0.002 mM
0.00046 - 0.04
fosfomycin
0.0004
thimerosal
Haemophilus influenzae
-
50 mM Tris-HCl, pH 7.8, compound displays an anti-MurA activity that is above 90% at a concentration below 0.002 mM
0.0007
thiram
Haemophilus influenzae
-
50 mM Tris-HCl, pH 7.8, compound displays an anti-MurA activity that is above 90% at a concentration below 0.002 mM
additional information
additional information
Bacillus anthracis
-
in wild-type background, IC50 of fosfomycin is 2,048 microg/ml. In a strain with antisense-based expression attenuation of the MurA1 gene encoding UDP-N-acetylglucosamine enolpyruvyl transferase, IC50 value is 126 microg/ml. Prsence of xylose further decreases the IC50 value to 18 microg/ml
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.1 - 0.12
-
partially purified enzyme
0.2
-
partially purified enzyme
0.27
-
purified enzyme
0.91
-
mutant enzyme C117D, at pH 6.0 and 25C
1.8
-
purified native enzyme
1.9
-
purified enzyme
4.7
-
wild type enzyme, at pH 7.8 and 25C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
assay at
additional information
-
determination of pKa value for Cys115: 8.3
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 10
-
recombinant enzyme, enzyme activity gradually decreases when the pH is below 5.5 or above 10.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Enterobacter cloacae subsp. cloacae (strain ATCC 13047 / DSM 30054 / NBRC 13535 / NCDC 279-56)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Streptococcus pneumoniae (strain Hungary19A-6)
Streptococcus pneumoniae serotype 2 (strain D39 / NCTC 7466)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio fischeri (strain MJ11)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34000
-
gel filtration
37000
-
nondenaturing PAGE
44700
calculated from the 422 amino acids, confirmed by SDS-PAGE
44800
MW deduced from amino acid sequence
45000
-
dynamic light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with UDP-N-acetylglucosamine and fosfomycin, sitting drop vapor diffusion method, using 25% (w/v) PEG 3350, 0.1 M bis-Tris pH 6.5
at 19C, from 10 mM MES, pH 6.4, 10% (w/v) polyethylene glycol 20000, in the presence of 5 mM UDP-N-acetyl-D-glucosamine and 5 mM phosphoenolpyruvate
-
crystallization of substrate-free enzyme, two-domain structure with an unusual fold, inside out alpha/beta barrel, which is built up from the 6fold repetititon of one folding unit, structure analysis
-
in complex with inhibitor T6361
-
in complex with substrates, hanging drop vapor diffusion method, using 0.1 M Bis-Tris (pH 5.5), 0.2 M NH4OAc, 25% (w/v) PEG 3350
-
mutant D305A, in presence of phosphoenolpyruvate and UDP-N-acetyl-D-glucosamine
-
X-ray structure analysis of ligated and unligated MurA
-
crystallization of the enzyme complexed with UDP-N-acetylglucosamine and fosfomycin, two-domain structure with the active site located between them, structure and substrate binding analysis
-
in complex with inhibitor cnicin and substrate UDP-N-acetylglucosamine, at 2.0 A resolution. The enzyme catalyzes the formation of a covalent adduct between cnicin and UDP-N-acetylglucosamine via an anti-Michael 1,3-addition of UDP-N-acetylglucosamine to an alpha,beta-unsaturated carbonyl function in cnicin thus forming a noncovalent suicide inhibitor
-
MurA is crystallized in the presence of sodium phosphite and UDP-N-acetylmuramic acid using the hanging drop vapor diffusion method, the MurA cocrystal structure with UDP-N-acetylmuramic acid and phosphite reveals a new staged MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site
-
in complex with UDP-N-acetylglucosamine and fosfomycin, to 2.3 A resolution. The active-site loop can adopt one of the three major conformations: a fully open conformation, a half-open conformation, and a closed conformation. Fosfomycin can bind without inducing a large change in the half-open conformation of the binary complex with UDP-N-acetylglucosamine
-
in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant, hanging drop vapour diffusion method
-
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
EDTA, DTT and dithioerythritol stabilize
-
no effect of 10% DMSO on the activity of enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, crude enzyme preparation, in presence of 4 mM DTT, loss of 84% activity within 2 weeks
-
-196C, 4 mM DTT, stable for up to 6 weeks, partially purified enzyme
-
-196C, slightly increased activity after storage of 2 weeks
-
-20C, loss of activity after 1 week
-
-70C, 4 mM DTT, 22% loss of activity in 2 weeks, partially purified enzyme
-
0C or -20C, 0.2 mg/ml DTT, 40% loss of activity after 2 weeks
-
4C, in 80% ammonium sulfate, loss of activity after 1 week
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, phenyl-Sepharose column chromatography, Q-Sepharose column chromatography, Superdex 200 gel filtration, and Sephadex G-25 gel filtration
-
by metal affinity chromatography with a nickel column
by using affinity chromatography
-
covalent adduct of enzyme and phosphoenolpyruvate
-
MurA purification is done by using self packed amylose resin column
native and recombinant from Escherichia coli
Ni-NTA agarose column chromatography and Superdex 75 gel filtration
Ni-NTA column chromatography and gel filtration
-
Ni-NTA resin chromatography, HiLoad XK 16 Superdex 200 prep-grade gel filtration, and Mono Q HR10/10 column chromatography
-
nickel affinity column chromatography
-
on to a HiTrap QP-Sepharose HP ion exchange column
-
purified protein has significant amounts of UDP-N-acteylmuramic acid bound
-
recombinant from Escherichia coli
-
recombinant from overexpresssing strain
-
recombinant wild-type and mutants from Escherichia coli
-
recombinant, 2 isoforms from Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and expression of MurA using pMAL expression system in frame with the fusion maltose binding protein in Escherichia coli BL21
coding region of Haemophilus influenzae MurA is cloned into the NdeI and XhoI sites of pET21a to generate an expression vector for MurA, the expression vector is transformed into Escherichia coli Rosetta2 (DE3), and the recombinant MurA is expressed
-
DNA and amino acid sequence determination and analysis, chromosome mapping at 69.3 min, overexpression in Escherichia coli strain JLM 16
DNA and amino acid sequence determination, functional overexpression in Escherichia coli strain JM105
-
expressed in an Enterococcus faecalis mutant lacking IreK (formerly PrkC), a key kinase required for cephalosporin resistance, and in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli
-
expressed in Escherichia coli BL21 Star (DE3) cells
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli DH5alpha cells
expressed in Escherichia coli STBL2-DE3 cells
-
expression from plasmid in Escherichia coli deletion mutant, functional complementation
expression in Escherichia coli
-
expression of wild-type and mutants in Escherichia coli
-
gene murA, DNA sequence analysis, expression in Escherichia coli under control of the arabinose-inducible, glucose-repressible ara promotor, functional complementation, necessary for viability, of a enzyme-deficient Escherichia coli murA null-mutant, impartment of fosfomycin resistance to the Escherichia coli cell
-
genes murA1 and murA2, DNA and amino acid sequence determination, overexpression of both isoforms in Escherichia coli BL21(DE3)
-
overexpression in Escherichia coli
-
overexpression in Escherichia coli with a 6-His tag
overexpression of wild type MurA
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the murA gene is cloned and overexpressed in Escherichia coli with a C-terminal 6-His tag; the murZ gene is cloned and overexpressed in Escherichia coli with a C-terminal 6-His tag
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C115D
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the mutant enzyme lacks the ability to react with phosphoenolpyruvate covalently
C251S
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site-directed mutagenesis, Cys251 is not involved in the catalysis, unaltered biochemical properties
C354S
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site-directed mutagenesis, Cys354 is not involved in the catalysis, unaltered biochemical properties
C381S
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site-directed mutagenesis, Cys381 is not involved in the catalysis, unaltered biochemical properties
D305C
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity
D305E
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, 0.1% activity compared to the wild-type
D305H
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115
K22V/R120K
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no residual activity, heat capacity changes are markedly redcued
K22V/R120V
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no residual activity
N23A
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site-directed mutagenesis, reduced activity, 20fold higher apparent dissociation constant for fosfomycin compared to wild-type
N23S
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site-directed mutagenesis, reduced activity, 200fold higher apparent dissociation constant for fosfomycin compared to wild-type
R120K
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less than 0.05% of wild-type activity, heat capacity changes are markedly redcued
C115S
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-
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C251S
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site-directed mutagenesis, Cys251 is not involved in the catalysis, unaltered biochemical properties
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C354S
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site-directed mutagenesis, Cys354 is not involved in the catalysis, unaltered biochemical properties
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C381S
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site-directed mutagenesis, Cys381 is not involved in the catalysis, unaltered biochemical properties
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D305A
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115
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D305C
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity
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D305E
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site-directed mutagenesis, weaker binding of UDP-GlcNAc, 0.1% activity compared to the wild-type
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N23A
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site-directed mutagenesis, reduced activity, 20fold higher apparent dissociation constant for fosfomycin compared to wild-type
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N23S
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site-directed mutagenesis, reduced activity, 200fold higher apparent dissociation constant for fosfomycin compared to wild-type
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C120S
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catalytically inactive
C115A
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site-directed mutagenesis, overexpression in Escherichia coli, no activity
C115E
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site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, enhanced pH-dependency of the reaction, low activity
C115N
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site-directed mutagenesis, overexpression in Escherichia coli, deamination of Asn115 to Asp115
C115S
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site-directed mutagenesis, overexpression in Escherichia coli, no activity
C115A
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site-directed mutagenesis, overexpression in Escherichia coli, no activity
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C115D
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site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, forms only the phospholactyl-enzyme intermediate adduct, but no UDP-GlcNAc-phosphoenolpyruvate, higher kcat than the wild-type at pH 7.0, enhanced pH-dependency of the reaction
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C115E
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site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, enhanced pH-dependency of the reaction, low activity
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C115N
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site-directed mutagenesis, overexpression in Escherichia coli, deamination of Asn115 to Asp115
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C115S
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site-directed mutagenesis, overexpression in Escherichia coli, no activity
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C117D
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the mutation shifts ithe optimum pH from 7.8 to 6.0. The mutant is not inhibited by 1 mM fosfomycin
C115D
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mutant protein is functional and resistant to fosfomycin and inhibitors 2-[4-(2-hydroxyethyl)piperazin-1-yl]-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one and2-(4-methylpiperazin-1-yl)-3,4-dihydronaphthalen-1(2H)-one
D120C
the mutant shows resistance against fosfomycin
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
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