Information on EC 1.14.20.1 - deacetoxycephalosporin-C synthase

Word Map on EC 1.14.20.1
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:


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

EC NUMBER
COMMENTARY hide
1.14.20.1
-
RECOMMENDED NAME
GeneOntology No.
deacetoxycephalosporin-C synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
deacetylcephalosporin C biosynthesis
-
-
Penicillin and cephalosporin biosynthesis
-
-
Metabolic pathways
-
-
Biosynthesis of antibiotics
-
-
SYSTEMATIC NAME
IUBMB Comments
penicillin-N,2-oxoglutarate:oxygen oxidoreductase (ring-expanding)
Forms part of the penicillin biosynthesis pathway (for pathway, click here).
CAS REGISTRY NUMBER
COMMENTARY hide
85746-10-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Cephalosporium acremonium, gene cefEF
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain NRRL3802
-
-
Manually annotated by BRENDA team
strain NRRL 3585
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
DAOCS is classified to the 2-oxoglutarate-Fe(II)-dioxygenase superfamily
metabolism
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
(2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(thiophen-2-ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid + 2-oxoglutarate + O2
(6R,7R)-3-methyl-8-oxo-7-[(thiophen-2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
3-exomethylenecephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
8% of the activity with cephalosporin N, recombinant enzyme
-
?
6-alpha-methylpenicillin N + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
low activity, hydroxylation reaction
-
-
ir
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
poor substrate
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
amoxicillin + 2-oxoglutarate + O2
?
show the reaction diagram
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
ampicillin + 2-oxo-4-methyl-pentanoic acid + O2
?
show the reaction diagram
-
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
ampicillin + 2-oxo-4-methylpentanoate + O2
cephalexin + succinate + CO2 + H2O
show the reaction diagram
-
wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids
-
?
ampicillin + 2-oxoglutarate + O2
?
show the reaction diagram
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
-
?
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
show the reaction diagram
ampicillin + 2-oxohexanoate + O2
cephalexin + ?
show the reaction diagram
-
wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids
-
-
?
ampicillin + 2-oxohexanoic acid + O2
?
show the reaction diagram
-
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
?
show the reaction diagram
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
cephalexin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
low activity, hydroxylation reaction
-
-
ir
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
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
-
-
-
-
?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
metampicillin + 2-oxoglutarate + O2
?
show the reaction diagram
metampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
N-((thiophen-2-yl)acetyl)-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-[(5R,6R)-3,3-dimethyl-2,7-dioxo-4-thia-1-azabicyclo[3.2.0]hept-6-yl]-2-(thiophen-2-yl)acetamide + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-acetyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-adipyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
N-butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-butyryldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-decanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-heptanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-hexanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-valeryldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin F + 2-oxoglutarate + O2
?
show the reaction diagram
-
-
-
?
penicillin G + 2-oxo-4-methyl-pentanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + 3-methylbutanoate + CO2 + H2O
show the reaction diagram
-
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
penicillin G + 2-oxo-4-methylpentanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
show the reaction diagram
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin G + 2-oxoglutarate + O2
?
show the reaction diagram
low catalytic effciency towards penicillin G
-
-
?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
penicillin G + 2-oxohexanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
show the reaction diagram
penicillin G + 2-oxohexanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + pentanoate + CO2 + H2O
show the reaction diagram
-
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
penicillin mX + 2-oxoglutarate + O2
7-[(3-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin mX + 2-oxoglutarate + O2
?
show the reaction diagram
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
penicillin V + 2-oxoglutarate + O2
?
show the reaction diagram
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
-
-
-
?
penicillin X + 2-oxoglutarate + O2
?
show the reaction diagram
-
-
-
?
phenethicillin + 2-oxoglutarate + O2
?
show the reaction diagram
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
-
?
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
phenyl-7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
show the reaction diagram
hydroxylation reaction
-
-
ir
ticarcillin + 2-oxoglutarate + O2
?
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
(2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(thiophen-2-ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid + 2-oxoglutarate + O2
(6R,7R)-3-methyl-8-oxo-7-[(thiophen-2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
amoxicillin + 2-oxoglutarate + O2
?
show the reaction diagram
ampicillin + 2-oxoglutarate + O2
?
show the reaction diagram
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
?
show the reaction diagram
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
show the reaction diagram
P18548
-
-
-
?
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
-
-
-
-
?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
metampicillin + 2-oxoglutarate + O2
?
show the reaction diagram
P18548
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
penicillin mX + 2-oxoglutarate + O2
7-[(3-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
show the reaction diagram
penicillin V + 2-oxoglutarate + O2
?
show the reaction diagram
P18548
-
-
-
?
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
show the reaction diagram
P18548
-
-
-
?
phenethicillin + 2-oxoglutarate + O2
?
show the reaction diagram
P18548
-
-
-
?
ticarcillin + 2-oxoglutarate + O2
?
show the reaction diagram
P18548
-
-
-
?
additional information
?
-
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
-
reversible, 0.04 mM, 90% inhibition
1,10-phenanthroline
-
-
2-oxobutyrate
-
0.1 mM, complete inhibition
3-exomethylenecephalosporin
-
strong, substrate for hydroxylation reaction of enzyme, EC 1.14.11.26
-
3-exomethylenecephalosporin C
-
0.28 mM, 71% inhibition of the formation of deacetoxycephalosporin C
3-oxoadipate
-
0.1 mM, 56% inhibition
5,5'-dithiobis-2-nitrobenzoic acid
ammonium hydrogencarbonate
-
100-500 mM, complete inactivation
ampicillin
-
; 2.8 mM, 13% inhibition of the formation of deacetoxycephalosporin C
bathophenanthroline
-
-
Cu2+
-
1 mM, complete inhibition
iodoacetic acid
N-ethylmaleimide
Ni2+
-
inhibition in decreasing order, Zn2+, Co2+, Ni2+
o-phenanthroline
p-hydroxymercuribenzoate
penicillin G
-
; 2.8 mM, 57% inhibition of the formation of deacetoxycephalosporin C
Penicillin V
-
; 2.8 mM, 47% inhibition of the formation of deacetoxycephalosporin C
additional information
-
when 2-oxoglutarate is added to the enzyme before the mixture of ATP, Fe2+ and ascobate activity is only about 40% of the reaction
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ammonium chloride
-
-
ammonium sulfate
-
-
ascorbate
bovine serum albumin
-
stimulates
-
catalase
-
stimulates
-
dithiothreitol
DTT
-
reducing agent stimulates DAOC synthase activity
Fe2+
requires the Fe2+ as a cofactor
glutathione
-
reduced
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003 - 0.022
2-oxoglutarate
3.28
acetyl-6-aminopenicillanic acid
wild type enzyme
1.3
adipyl 6-aminopenicillanic acid
-
-
0.05 - 24
ampicillin
0.028
deacetoxycephalosporin C
-
-
0.003
oxoglutarate
-
-
0.014 - 48.1
penicillin G
0.004 - 0.295
Penicillin N
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.06
acetyl-6-aminopenicillanic acid
-
0.073
adipyl 6-aminopenicillanic acid
-
-
0.005 - 0.122
ampicillin
0.001 - 0.668
penicillin G
0.02 - 0.42
Penicillin N
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.003 - 0.736
penicillin G
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.034
3-exomethylenecephalosporin C
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.013
recombinant enzyme, substrate amoxicillin
0.014
purified recombinant wild-type and truncated mutant enzyme, substrate metampicillin
0.015
-
recombinant enzyme, substrate metampicillin
0.023
purified recombinant truncated mutant enzyme, substrate carbenicillin
0.037
-
recombinant enzyme, substrate carbenicillin
0.039
purified recombinant wild-type and truncated mutant enzyme, substrate ampicillin, determined by bioassay
0.051
recombinant enzyme, substrate penicillin V
0.071
purified recombinant truncated mutant enzyme, substrate phenethicillin
0.074
purified recombinant wild-type enzyme, substrate phenethicillin
0.128
recombinant enzyme, substrate carbenicillin
0.133
purified recombinant wild-type enzyme, substrate ampicillin, determined by HPLC
0.156
purified recombinant wild-type enzyme, substrate 6-alpha-methylpenicillin N, substrate conversion
0.166
-
native enzyme
0.178
purified recombinant truncated mutant enzyme, substrate ampicillin, determined by HPLC
0.203
-
recombinant enzyme, substrate ampicillin, determined by bioassay
0.237
purified recombinant truncated mutant enzyme, substrate penicillin G
0.24
purified recombinant wild-type enzyme, substrate penicillin G
0.36
-
recombinant enzyme, substrate penicillin G
0.411
-
recombinant enzyme, substrate phenethicillin
0.432
-
recombinant enzyme
0.458
recombinant enzyme, substrate ampicillin, determined by bioassay
0.76
-
substrate carbenicillin, wild-type, pH 7.4, 30°C
0.827
-
pH 7.5, 30°C
0.932
recombinant enzyme, substrate ampicillin; recombinant enzyme, substrate ampicillin, determined by HPLC
1.042
recombinant enzyme, substrate penicillin G
1.551
recombinant enzyme, substrate phenethicillin
2.221
-
recombinant enzyme, substrate ampicillin, determined by HPLC
3.57
-
substrate phenethicillin, wild-type, pH 7.4, 30°C
5.67
-
substrate ampicillin, wild-type, pH 7.4, 30°C
6.41
-
substrate ampicillin, wild-type, 30°C
6.72
-
substrate penicillin G, wild-type, pH 7.4, 30°C
7.23
-
substrate ampicillin, mutant R306M, 30°C
9.11
-
substrate ampicillin, mutant R306I, 30°
10.9
-
substrate ampicillin, mutant R306L, 30°C
11.89
-
recombinant mutant S261I, pH not specified in the publication, 30°C
13.44
-
recombinant mutant C37S, pH not specified in the publication, 30°C
16.87
-
recombinant mutant T42A, pH not specified in the publication, 30°C
20.03
-
recombinant mutant A61E, pH not specified in the publication, 30°C
22.06
-
recombinant mutant S261V, pH not specified in the publication, 30°C
30.86
-
recombinant mutant S261L, pH not specified in the publication, 30°C
31.38
-
recombinant mutant Y184L, pH not specified in the publication, 30°C
33.51
-
recombinant mutant Y184M, pH not specified in the publication, 30°C
36.12
-
recombinant mutant Y184I, pH not specified in the publication, 30°C
38.5
-
pH 7.4, 27°C
49.12
-
recombinant mutant S59T, pH not specified in the publication, 30°C
56.84
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
66
-
recombinant enzyme
71.7
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
80.68
-
recombinant mutant T213V, pH not specified in the publication, 30°C
90.04
-
recombinant mutant S261M, pH not specified in the publication, 30°C
98
-
recombinant mutant S261A, pH not specified in the publication, 30°C
160
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
190
-
purified recombinant wild-type
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
-
assay at, substrate penicillin G
7
-
; in piperazineethanesulfonic acid buffer, recombinant enzyme
7.4
-
native enzyme
7.5 - 7.8
-
expandase reaction, EC 1.14.20.1
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26 - 34
-
expandase reaction, EC 1.14.20.1
36
-
; native and recombinant enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 40
-
20°C: 15% of maximal activity, 35°C: 15% of maximal activity
20 - 35
-
20°C: 90% of maximal activity, 35°C: 90% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3
-
isoelectric focusing
6.1
-
isoelectric focusing
6.3
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
Streptomyces clavuligerus (strain ATCC 27064 / DSM 738 / JCM 4710 / NBRC 13307 / NCIMB 12785 / NRRL 3585 / VKM Ac-602)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
-
gel filtration
28000
-
1 * 28000, SDS-PAGE
29500
-
gel filtration
30966
-
3 * 30966, DAOCS is in equilibrium with a trimeric form which is the form that crystallizes
34500
-
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE
34519
-
x * 34519, calculation from nucleotide sequence; x * 36000, SDS-PAGE, x * 34519, calculated
34554
-
x * 34554, in solution the monomeric apoenzyme is in equilibrium with atrimeric form, electrospray mass spectrometry
35000
-
gel filtration
36000
-
x * 36000, SDS-PAGE; x * 36000, SDS-PAGE, x * 34519, calculated
36348
x * 36348, wild-type enzyme, electrospray mass spectrometry
36642
-
x * 40000, SDS-PAGE, x * 36642, calculated
41000
-
1 * 41000, SDS-PAGE
43000
-
gel filtration
62000
x * 60000, recombinant truncated mutant enzyme, SDS-PAGE, x * 62000, recombinant wild-type enzyme
additional information
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
trimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the DAOC synthase is in trimeric form, C-terminal end of the protein is responsible for the oligomerization process, addition of Fe2+ or 2-oxoglutarate shifts the equilibrium toward the monomeric form that appears to be the active form in solution
-
; as apoprotein, 1.3 A resolution; as apoprotein with N-terminal His tag, 2.3 A resolution; crystallization of mutant delta R306, with Fe2+ and 2-oxoglutarate, 2.1 A resolution; crystallization of mutant delta R307A, with Fe2+, succinate and CO2, 1.96 A resolution; crystallization with 5-hydroxy-4-ketovaleric acid, 1.53 A resolution; crystallization with Fe2+, 1.5 A resolution; crystallization with Fe2+ and 2-oxoglutarate, 1.5 A resolution, the crystal structure of scDAOCS complexed with 2-oxoglutarate reveals that the 5-carboxylate of 2-oxoglutarate is stabilized by electrostatic interaction with the side chain of R258; crystallization with Fe(II), 2-oxoglutarate and ampicillin, 1.5 A resolution; crystallization with Fe(II), 2-oxoglutarate and penicillin G, 1.7 A resolution; crystallization with Fe(II) and deacetoxycephalosporin C, 1.7 A resolution; crystallization with Fe(II) and penicillin G, 1.6 A resolution; crystallization with Fe(II) and succinate , 1.5 A resolution; R258Q mutant, crystallization with Fe(II) and alpha-keto-beta-methylbutanoate, 1.5 and 1.6 A resolution; with N-terminal His tag and Fe2+, 2.51 A resolution; with N-terminal His tag, crystallization with ampicillin and Fe2+, 2.7 A resolution; with N-terminal His tag, crystallization with deacetoxycephalosporin C and Fe2+, 3.0 A resolution
crystallization of wild-type enzyme and of the DELTAR306 mutant complexed with iron(II) and 2-oxoglutarate to 2.1 A and the DELTAR306A mutant complexed with iron(II), succinate and unhydrated carbon dioxide to 1.96 A
-
hanging drop method, recombinant enzyme expressed in Escherichia coli
-
quantum mechanical calculations of the first part of the reaction based on the high-resolution structures of the active site and its complexes with ligands
-
recombinant enzyme expressed in Escherichia coli, high-resolution structures for apoenzyme, the enzyme complexed with Fe(II), and with Fe(II) and 2-oxoglutarate
-
recombinant N-terminally His-tagged wild-type and C-terminally truncated mutant enzymes, free enzyme, or complexing with Fe2+, or Fe2+/ampicillin, hanging drop vapour diffusion method, 4°C, precipitant solution: 100 mM HEPES-NaOH, pH 8.0, 0.9-1.1 M ammonium sulfate, the reservoir solution is covered with oil to retard the evaporation, cryoprotection by 30% v/v ethylene glycol in precipitant solution, X-ray structure determination and analysis at 2.3 A resolution, molecular modeling
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
-
; 4°C, 1 h, stable, native enzyme
440380
7
-
4°C, 120 h, 80% loss of activity
440378
8
-
4°C, 120 h, 70% loss of activity
440378
9
-
4°C, 120 h, 35% loss of activity
440378
10
-
enzyme has similar stability at 4°C and at 25°C
440375
11
-
enzyme is more stable at 4°C than at 25°C
440375
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
60 min, stable
30
-
60 min, 25% loss of activity
40
-
10 min, 50% loss of activity; stable up to
65
-
60 min, complete inactivation
additional information
-
at pH 10 enzyme has similar stability at 4°C and at 25°C. At pH 11 the enzyme is more stable at 4°C than at 25°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
increasing ionic strength to 100 decreases stability of the enzyme
-
purified enzyme is stable when flash-frozen and stored at -80°C
-
sodium phosphate and Bis/Tris buffers are inhibitory
-
the enzyme is very unstable and can be partially stabilized in 25 mM Tris-HCl, pH 9.0, in the presence of 0.1 mM DTT
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
-
purification is achieved by an anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogenous unfolded protein into the active enzyme is a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, presence of dithiothreitol, stable for 4 weeks
-
4°C, presence of dithiothreitol, half-life is 48 h
-
purified enzyme is stable when flash-frozen and stored at -80°C
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; affinity chromatography
anion-exchange and gel filtration
-
efficient large-scale purification; recombinant enzyme expressed in Escherichia coli
-
expression in Escherichia coli, overexpression as an insoluble and inactive enzyme, elaboration of refolding scheme resulting in highliy active and moderately stable enzyme
-
native enzyme, partial, recombinant enzyme, to near homogenity; recombinant enzyme expressed in Escherichia coli
-
purification of partially folded recombinant expandase/hydroxylase from insoluble granules of recombinant Escherichia coli
-
purification of refolding-competent recombinant enzyme
-
recombinant enzyme expressed in Escherichia coli
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
recombinant N-terminally His-tagged wild-type and C-terminally truncated mutant enzymes from Escherichia coli strain BL21(DE3)
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography and gel filtration; recombinant wild-type enzyme and mutant enzymes R258K, R258H, R258A, R258L and R258F
-
recombinant wild-type and mutant enzymes from Escherichia coli strain ESS by ion exchange chromatography and gel filtration
-
soluble recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 by anion exchange chromatography and gel filtration
-
wild-type and mutant enzymes R74I, R74Q, R75I/D270G, R75Q, R306L and R307Q
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and heterologous expression in Escherichia coli, Streptomyces lividans, Penicillium chrysogenum, Pseudomonas putida and Pichia pastoris, using of commonly used glutathione S-transferase to express DAOCS as a fusion protein; expressed in Escherichia coli, Streptomyces lividans, Penicillium chrysogenum, Pseudomonas putida, and Pichia pastoris; the attempt to improve the activity of DAOCS by the directed evolution approach is probably the construction of hybrid DAOCS of Streptomyces clavuligerus and Nocardia lactamdurans using in-vivo homologous recombination
DNA and amino acid sequence determination and analysis, expression as GST-fusion protein in Escherichia coli strain BL21(DE3)
DNA and amino acid sequence determination and analysis, expression of the wild-type enzyme and a C-terminally truncated mutant enzyme lacking 20 amino acids as GST-fusion proteins in Escherichia coli strain BL21(DE3)
expressed in Escherichia coli
expression as glutathione S-transferase fusion protein in Echerichia coli
-
expression at high levels in Escherichia coli under control of the trc promoter
-
expression in Escherichia coli
-
expression of wild-type and C-terminally truncated mutant enzymes as N-terminally His-tagged proteins in Escherichia coli strain BL21(DE3)
-
expression of wild-type and mutant enzymes at high levels in Escherichia coli
-
expression of wild-type and mutant enzymes in Escherichia coli strain BL21
-
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in strain XL-1 Blue
expression of wild-type and mutant enzymes in Escherichia coli strain ESS
-
overexpression as an insoluble and inactive enzyme in granules of recombinant Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
DELTA310/M306I
site-directed mutagenesis, mutant selectively catalyzed the ring expansion with kinetics similar to the wild-type enzyme
DELTA310/N305L/M306I
site-directed mutagenesis, mutant selectively catalyzed the ring expansion, and shows a lower Km value than the wild-type enzyme and about 3fold improved kinetic parameters
M306I
hydroxylation reaction of deacetoxycephalosporin C, E C1.14.11.26, is abolished, 59% of wild-type ring expansion activity; site-directed mutagenesis, mutant shows no hydroxylation of desacetylcephalosporin C
R307L
-
activities comparable to wild-type
R308A
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308C
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308D
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308E
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308F
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308G
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308H
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308I
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme. The mutant shows the highest reactivity for penicillin G, with 3fold increase in kcat/Km ratio and 7fold increase in relative activity
R308K
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308M
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308N
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308P
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308Q
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308S
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308T
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme
R308V
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme
R308W
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
R308Y
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme
W82A
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; site-directed mutagenesis, mutant shows reduced ring expansion activity with artificial substrate penicillin G
W82S
44% of wild-type ring expansion activity, 18% of wild-type hydroxylation activity
A106T
-
80% relative activity with 1 mM substrate and 170% relative activity with 10 mM penicillin G compared to wild-type activity detected with 1 mM penicillin G, 1.5fold reduction in Km, penicillin G as substrate
A106T/C155Y
-
epPCR random mutagenesis
A106T/M188V
-
epPCR random mutagenesis
A11V/T91A/C281Y/I305L
-
220% relative activity with 1 mM and 200% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
A61E
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
C155Y/Y184H/V275I/C281Y
C281F
-
substrate ampicillin, 191%, penicillin G, 264%, phenethicillin, 300%, and carbenicillin, 518% of wild-type activity
C281R
-
activity similar to wild-type enzyme
C281Y/I305M
C281Y/N304A
-
substrate ampicillin, 294%, penicillin G, 383%, phenethicillin, 714%, and carbenicillin, 911% of wild-type activity
C281Y/N304K
-
substrate ampicillin, 376%, penicillin G, 428%, phenethicillin, 948%, and carbenicillin, 1180% of wild-type activity
C281Y/N304L
-
substrate ampicillin, 330%, penicillin G, 211%, phenethicillin, 471%, and carbenicillin, 1001% of wild-type activity
C281Y/N304M
-
substrate ampicillin, 225%, penicillin G, 277%, phenethicillin, 428%, and carbenicillin, 485% of wild-type activity
C281Y/N304R
C281Y/R306L
-
substrate ampicillin, 394%, penicillin G, 191%, phenethicillin, 555%, and carbenicillin, 1010% of wild-type activity
C281Y/R307L
-
substrate ampicillin, 274%, penicillin G, 334%, phenethicillin, 520%, and carbenicillin, 786% of wild-type activity
C37S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
D107G
-
activity similar to wild-type enzyme
D107G/L277Q
-
epPCR random mutagenesis
D185L
-
no detectable ring expansion activity
D53H/C281Y
-
epPCR random mutagenesis
DELTAI305-310
-
mutant truncated by six residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme
DELTAK310-314
-
mutant truncated by five residues, increased turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme, lower Km-value for penicillin G compared to wild-type enzyme, slightly higher turnover number for penicillin G compared to wild-type enzyme
DELTAR306-310
-
mutant truncated by five residues, reduced turnover number for penicillin N compared to wild-type enzyme, lower KM-value for penicillin N compared to wild-type enzyme
DELTAR307-310
-
mutant in which residues 307-310 are excised, reduced turnover number for penicillin N compared to wild-type enzyme, higher KM-value for penicillin N compared to wild-type enzyme, increased KM-value for penicillin G compared to wild-type enzyme, increased turnover-number for penicillin G compared to wild-type enzyme
E144K/M188I/A198T/V275I/C281Y
-
160% relative activity with 1 mM and 200% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
G300V
-
410% relative activity with 1 mM and 370% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G; substrate inhibition, epPCR random mutagenesis
H183L
-
no detectable ring expansion activity
H243L
-
no detectable ring expansion activity
I305M/S261M
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V/M73T