Information on EC 1.14.11.26 - deacetoxycephalosporin-C hydroxylase

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

EC NUMBER
COMMENTARY hide
1.14.11.26
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RECOMMENDED NAME
GeneOntology No.
deacetoxycephalosporin-C hydroxylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
deacetoxycephalosporin C + 2-oxoglutarate + O2 = deacetylcephalosporin C + succinate + CO2
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
deacetylcephalosporin C biosynthesis
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Penicillin and cephalosporin biosynthesis
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Metabolic pathways
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Biosynthesis of antibiotics
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SYSTEMATIC NAME
IUBMB Comments
deacetoxycephalosporin-C,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating)
Requires iron(II). The enzyme can also use 3-exomethylenecephalosporin C as a substrate to form deacetoxycephalosporin C, although more slowly [2]. In Acremonium chrysogenum, the enzyme forms part of a bifunctional protein along with EC 1.14.20.1, deactoxycephalosporin-C synthase. It is a separate enzyme in Streptomyces clavuligerus.
CAS REGISTRY NUMBER
COMMENTARY hide
69772-89-0
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85746-10-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Cephalosporium acremonium, gene cefEF
SwissProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3'-methylcephem + 2-oxoglutarate + O2
3'-hydroxymethylcephem + succinate + CO2
show the reaction diagram
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-
-
-
?
3-exomethylenecephalosporin + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
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-
-
-
?
3-exomethylenecephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
show the reaction diagram
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-
-
-
?
3-oxomethylenecephalosporin C + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2
show the reaction diagram
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37% of activty with deacetoxycephalosporin C
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-
?
7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
7-aminodeacetylcephalosporin + succinate + CO2
show the reaction diagram
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
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-
-
-
?
cephalexin + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
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-
-
-
?
cephalosporin G + 2-oxoglutarate + O2
deacetylcephalosporin G + succinate + CO2
show the reaction diagram
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
show the reaction diagram
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
show the reaction diagram
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-
-
-
?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
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-
-
-
?
penicillin N + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
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-
-
-
?
penicillin N + 2-oxoglutarate + O2
succinate + CO2 + ?
show the reaction diagram
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-
-
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?
phenylacetyl-7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetylcephalosporin + succinate + CO2
show the reaction diagram
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
3-exomethylenecephalosporin + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
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-
-
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?
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
show the reaction diagram
penicillin N + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
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-
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S,4S,6R,7R)-1-aza-7-((5R)-5-carboxypentanamidol)-4-methyl-8-oxo-5-thiatricyclo-(4,2,0,0)octane-2-carboxylate
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reversible, 0.04 mM, 90% inhibition
5,5'-dithiobis-2-nitrobenzoic acid
ammonium hydrogencarbonate
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100-500 mM, complete inactivation
bathophenanthroline
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-
Co2+
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inhibition in decreasing order, Zn2+, Co2+, Ni2+
iodoacetic acid
N-ethylmaleimide
Ni2+
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inhibition in decreasing order, Zn2+, Co2+, Ni2+
o-phenanthroline
p-hydroxymercuribenzoate
Penicillin N
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1 mM, 62% inhibition, competitive with deacetoxycephalosporin C
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonium chloride
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ammonium sulfate
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ascorbate
dithiothreitol
glutathione
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reduced
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.022
2-oxoglutarate
2.58 - 9.2
cephalosporin G
0.02 - 0.059
deacetoxycephalosporin C
0.02
Fe2+
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0.19 - 2.58
penicillin G
0.004 - 0.295
Penicillin N
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.055 - 2.302
cephalosporin G
0.0315 - 0.1458
penicillin G
0.048 - 0.366
Penicillin N
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.021 - 0.43
cephalosporin G
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12
Penicillin N
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pH 7.3, 29C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.127
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pH 7.5, 36C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
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hydroxylation reaction, EC 1.14.11.26
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3
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isoelectric focusing
6.1
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isoelectric focusing
6.3
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isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26200
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gel filtration
34366
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x * 34366, calculated
35000
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gel filtration
36642
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x * 40000, SDS-PAGE, x * 36642, calculated
41000
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1 * 41000, SDS-PAGE
43000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 41000, SDS-PAGE
additional information
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structural model of enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
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440380
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
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stable up to
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
sodium phosphate and Bis/Tris buffers are inhibitory
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, presence of dithiothreitol, stable for 4 weeks
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4C, presence of dithiothreitol, half-life is 48 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion-exchange chromatography and gel filtration
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DAOC hydroxylase of Streptomyces clavuligerus is purified to near homogeneity by anion-exchange chromatography, ammonium sulphate precipitation and gel filtration
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expression in Escherichia coli, overexpression as an insoluble and inactive enzyme, elaboration of refolding scheme resulting in highly active and moderately stable enzyme
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native enzyme, partial, recombinant enzyme, to near homogenity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli Tuner(DE3) and BL21(DE3) cells
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expression in Escherichia coli
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expression in Streptomyces lividans
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geen cefF, recombinant exression of cefF library mutants, site-directed mutants, and wild-type enzyme in Escherichia coli strain BL21(DE3)
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gene cefF, subcloning in Escherichia coli, functional expression in Acremonium chrysogenum strain CGMCC3.3795 via Agrobacterium tumefaciens-mediated transformation under the promoter of pcbC, leading to a reduction of the content of deacetoxycephalosporin C in the cephalosporin C fermentation broth, quantitative PCR expression analysis
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
M306I
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hydroxylation reaction of deacetoxycephalosporin C, EC 1.14.11.26, is abolished, 59% of wild-type ring expansion activity
R308L
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improved ability to convert penicillin analogs in ring expansion reaction of EC 1.14.20.1
W82A
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5.5% of wild-type ring expansion activity, 71% of wild-type hydroxylation activity; ring expansion reaction of EC 1.14.20.1 is reduced
W82S
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44% of wild-type ring expansion activity, 18% of wild-type hydroxylation activity
A106T
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80% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
A177V
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
A311V
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random mutagenesis
C155Y
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90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
C155Y/Y184H/V275I/C281Y
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580% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
C281Y
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200% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
E16G/T90A/T304A
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme; site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
E209Q
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
E82D
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
F267L
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random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
G300V
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410% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
G79E
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90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
H244Q
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140% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
I193V
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random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
I305L
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230% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
I305M
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380% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
L236V
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
L277Q
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270% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
M184I
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
M188I
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90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
M188V
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150% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
M229V
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random mutagenesis
M73T
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180% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
N304K
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220% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
P186L
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random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
P72L
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random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
R182S
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
R182W
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random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
S251F
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
S260G
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T273A
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random mutagenesis
T90A
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random mutagenesis
T90A/A311V
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/M184I/I193V/F267L
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/R182S
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/S251F
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/S260G
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/F267L
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/A241V/V307A
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/N313D
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G/A241V/V307A
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/A241V/V307A
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/G255D/A280S
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/V226I
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site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/A40V/M229I/T273A/V221P
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/F195L
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/P7L/A237V
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/P7L/T273A
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/R250L
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/V206I/A210V
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T90A/P72L/A311V/V206I/A210V/T273A
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
T91A
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110% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
V150A
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random mutagenesis
V171L
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random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V171M
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V221A
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random mutagenesis
V221H
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V221P
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site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V221T
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random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V249I
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
V275I
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270% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
V275I/I305M
-
500% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
Y184H
-
200% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate
Y38C
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random mutagenesis
A177V
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
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E82D
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random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme
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T90A
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random mutagenesis
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V221A
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random mutagenesis
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additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
enzyme inactivated by 5,5-dithiobis-2-nitrobenzoic acid, complete reactivation by dithiothreitol
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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production of beta-lactam antibiotics
synthesis
-
expression in Escherichia coli, overexpression as an insoluble and inactive enzyme, elaboration of refolding scheme resulting in highly active and moderately stable enzyme