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Enzyme Nomenclature
Information on EC 1.14.20.1 - deacetoxycephalosporin-C synthase
Word Map on EC 1.14.20.1
- 1.14.20.1
- streptomyces
- clavuligerus
- isopenicillin
- acremonium
-
cephamycins
-
epimerase
- chrysogenum
- cephalosporium
-
penicillium
- alpha-ketoglutarate
- beta-lactams
-
ring-expansion
- ampicillin
-
2-oxoglutarate-dependent
-
cephem
- lactamdurans
-
7-aminodeacetoxycephalosporanic
- carbenicillin
- adipate
-
ironii
- medicine
- biotechnology
- synthesis
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
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EC NUMBER 
COMMENTARY 
1.14.20.1
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RECOMMENDED NAME
GeneOntology No.
deacetoxycephalosporin-C synthase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
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penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
bifunctional enzyme, residues W82, N305 and M306 are involved in catalysis, overview
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
reaction mechanism
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penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
presence of a stable iron-peroxo intermediate in equilibrium with a more reactive ferryl species. Formation of CO2 as a leaving group by decarboxylation of 2-oxoglutarate. Substitution of CO2 by the penicillin substrate triggers the oxidation reaction in a booby-trap-like mechanism
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydroxylation
oxidation
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-
redox reaction
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-
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reduction
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
penicillin-N,2-oxoglutarate:oxygen oxidoreductase (ring-expanding)
Forms part of the penicillin biosynthesis pathway (for pathway, click here).
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CAS REGISTRY NUMBER
COMMENTARY
85746-10-7
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Cephalosporium acremonium, gene cefEF
SwissProt
bifunctional enzyme deacetoxycephalosporin C synthetase/hydroxylase
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catalyzes both reaction of EC 1.14.20.1 and of EC 1.14.11.26
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enzyme catalyzes both reaction of EC 1.14.20.1 and of EC 1.14.11.26
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recombinant enzyme, catalyzes both reaction of EC 1.14.20.1 and of EC 1.14.11.26
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SwissProt
; native enzyme and recombinant enzyme expressed in Escherichia coli, enzyme catalyzes both reaction of EC 1.14.20.1 and of EC 1.14.11.26
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also has activity of EC 1.14.11.26, deacetoxycephalosporin hydroxylase
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
DAOCS is classified to the 2-oxoglutarate-Fe(II)-dioxygenase superfamily
metabolism
additional information
metabolism
the enzyme from Acremonium chrysogenum is bifunctional and catalyzes both the synthesis of deacetoxycephalosporin C from penicillin N, EC 1.14.20.1, as well as the hydroxylation of deacetoxycephalosporin C to deacetylcephalosporin C, EC 1.14.11.26. The activities are located on two different domains
metabolism
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the enzyme from Acremonium chrysogenum is bifunctional and catalyzes both the synthesis of deacetoxycephalosporin C from penicillin N, EC 1.14.20.1, as well as the hydroxylation of deacetoxycephalosporin C to deacetylcephalosporin C, EC 1.14.11.26. The activities are located on two different domains
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additional information
structure simulations, docking, and modeling, active site prediction, residues R162, F164, M180, L204, V245, V262, F264, and I305 are involved in 2-oxoglutarate binding, overview
additional information
additional recombinant expression of gene cefF from Streptomyces clavuligerus in Acremonium chrysogenum strain CGMCC3.3795 leads to a reduction of the content of deacetoxycephalosporin C in the cephalosporin C fermentation broth, quantitative PCR expression analysis, overview
additional information
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additional recombinant expression of gene cefF from Streptomyces clavuligerus in Acremonium chrysogenum strain CGMCC3.3795 leads to a reduction of the content of deacetoxycephalosporin C in the cephalosporin C fermentation broth, quantitative PCR expression analysis, overview
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(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
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-
-
?
3-exomethylenecephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
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8% of the activity with cephalosporin N, recombinant enzyme
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?
7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
low activity, hydroxylation reaction
-
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ir
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
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-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
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-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
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poor substrate
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?
ampicillin + 2-oxo-4-methyl-pentanoic acid + O2
?
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mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
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ir
ampicillin + 2-oxo-4-methylpentanoate + O2
cephalexin + succinate + CO2 + H2O
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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
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?
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2
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?
ampicillin + 2-oxohexanoate + O2
cephalexin + ?
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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
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?
ampicillin + 2-oxohexanoic acid + O2
?
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mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
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ir
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
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-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2
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-
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?
cephalexin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
low activity, hydroxylation reaction
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ir
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
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-
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?
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
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?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
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-
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?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
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?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
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-
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?
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
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-
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?
N-acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-acetyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-adipyldeacetoxycephalosporin C + succinate + CO2 + H2O
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-
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?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
N-butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-butyryldeacetoxycephalosporin C + succinate + CO2 + H2O
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-
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?
N-decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-decanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-heptanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-hexanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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-
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?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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-
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?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-valeryldeacetoxycephalosporin C + succinate + CO2 + H2O
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-
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?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
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?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
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-
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?
penicillin G + 2-oxo-4-methyl-pentanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + 3-methylbutanoate + CO2 + H2O
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mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
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ir
penicillin G + 2-oxo-4-methylpentanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
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?
penicillin G + 2-oxoglutarate + O2
?
low catalytic effciency towards penicillin G
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?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2
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?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
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?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
penicillin G + 2-oxohexanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + pentanoate + CO2 + H2O
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mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
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ir
penicillin mX + 2-oxoglutarate + O2
7-[(3-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
-
-
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?
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
-
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?
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2
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-
-
-
?
phenyl-7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
hydroxylation reaction
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ir
6-alpha-methylpenicillin N + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
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?
6-alpha-methylpenicillin N + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
best substrate in the ring expansion reaction
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ir
6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
low activity, ring expansion reaction
-
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ir
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
ring expansion reaction
-
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ir
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
ring expansion reaction
-
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ir
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
low activity, ring expansion reaction
-
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ir
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
mutant R308L, 100% of activity, compared to wild-type
-
-
?
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
ring expansion reaction
-
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ir
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
-
mutant N305L, 222%, mutant R307L, 100%, mutant R308L, 311% of activity, compared to wild-type
-
-
?
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
-
-
-
?
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
-
-
-
-
?
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
-
no activity
-
-
-
ampicillin + 2-oxoglutarate + O2
cephalexin + succinate + CO2 + H2O
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
mutant N305L, 178%, mutant R307L, 93%, mutant R308L, 194% of activity, compared to wild-type
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
-
-
-
ir
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
-
-
-
ir
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
the penicillin /cephem substrate are bound by residues R161 and R163
-
-
ir
metampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
metampicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxo-4-methylpentanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
-
-
?
penicillin G + 2-oxo-4-methylpentanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
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
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
mutant N305L, 163%, mutant R307L, 100%, mutant R308L, 516% of activity, compared to wild-type
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
no activity
-
-
-
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
no activity
-
-
-
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
ir
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
the penicillin /cephem substrates are bound by residues R161 and R163
-
-
ir
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
ir
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
product is the precursor of 7-aminodeacetoxycephalosporanic acid, which is used in industrial applications
-
ir
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
ir
penicillin G + 2-oxohexanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
-
-
?
penicillin G + 2-oxohexanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
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
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
almost absolute requirement for 2-oxoglutarate
-
-
-
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
bifunctional enzyme: penicillin N expandase/DAOC hydroxylase
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
2-oxoglutarate is required, residues M181, R259, and S261 might be responsible for binding of 2-oxoglutarate, the penicillin /cephem substrate are bound by residues R161 and R163
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
ring expansion of penicillin N to a six member cephem ring, the natural enzyme is only able to use 2-oxoglutarate but not 2-oxobutyrate, 3-oxoadipate, 2-oxohexanoic acid or 2-oxo-4-methylpentanoic acid as cosubstrates
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
oxoglutarate analogs are not used as substrate
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
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
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
separate genes encode penicillin N expandase and DAOC hydroxylase
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
the enzyme catalyzes the committed step in the biosynthesis of cephalosporin antibiotics
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
committed step in biosynthesis of cephamycin C
-
-
-
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme is involved in catalyzing the biosynthesis of cephalosporins and cephamycin
-
-
-
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
first step in cephamycin C biosynthetic pathway
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
oxidative ring expansion via reactive iron-oxygen intermediate
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
specific for 2-oxoglutarate, which cannot be substituted by other 2-oxoacids e.g. 2-oxohexanoic acid, or 2-oxo-4-methyl-pentanoic acid
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
about 3%, compared to activity of EC 1.14.11.26, deacetoxycephalosporin hydroxylase
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?, ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
first step in cephamycin C biosynthetic pathway
-
-
ir
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
low activity, ring expansion reaction
-
-
ir
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
mutant N305L, 100%, mutant R307L, 93%, mutant R308L, 170% of activity, compared to wild-type
-
-
?
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
no activity
-
-
-
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
?
additional information
?
-
-
the enzyme catalyzes the first and second step in the cephamycin C biosynthetic pathway
-
-
-
additional information
?
-
the enzyme catalyzes the first and second step in the cephamycin C biosynthetic pathway
-
-
-
additional information
?
-
-
deacetoxycephem substrate specificity for the hydroxylation reaction, penam substrate specificity of the bifunctional enzyme for the ring-expansion reation, overview
-
-
-
additional information
?
-
deacetoxycephem substrate specificity for the hydroxylation reaction, penam substrate specificity of the bifunctional enzyme for the ring-expansion reation, overview
-
-
-
additional information
?
-
-
substrate specificity, oxacillin, penicillin V, and amoxicillin are no substrate
-
-
-
additional information
?
-
substrate specificity, oxacillin, penicillin V, and amoxicillin are no substrate
-
-
-
additional information
?
-
-
no substrate: pyruvate, 2-oxoadipate
-
-
-
additional information
?
-
-
a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C
-
-
-
additional information
?
-
-
role of residue R308 in controlling substrate selectivity, substrate specificity of wild-type and mutant enzymes, overview
-
-
-
additional information
?
-
-
no activity with isopenicillin N or 6-aminopenicillanic acid
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
-
substrate specificity, amoxicillin, penicillin V, and oxacillin are no substrate
-
-
-
additional information
?
-
-
has no hydroxylase activity of EC 1.14.11.26
-
-
-
additional information
?
-
-
no substrate: isopenicillin N, penicillin G, penicillin V, ampicillin, 6-aminopenicillanic acid
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
enzyme is able to convert a wide range of penicillin substrates differing in their side chains, it is a member of 2-oxoglutarate-dependent dioxygenase protein family, the mechanism involves initial activation of the iron centre by 2-oxoglutarate binding followed by oxidative decarboxylation of 2-oxoglutarate to generate oxidizing ferryl species with succinate and carbon dioxide as by-products, succinate remains bound and stabilizes the reactive iron species until the substrate enters the active site, binding of penicillin to the reactive iron species via the sulfur group subsequently expels succinate and leads to oxidative ring expansion of the substrate
-
-
-
additional information
?
-
-
Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G
-
-
-
additional information
?
-
-
Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
enzyme is able to convert a wide range of penicillin substrates differing in their side chains, it is a member of 2-oxoglutarate-dependent dioxygenase protein family, the mechanism involves initial activation of the iron centre by 2-oxoglutarate binding followed by oxidative decarboxylation of 2-oxoglutarate to generate oxidizing ferryl species with succinate and carbon dioxide as by-products, succinate remains bound and stabilizes the reactive iron species until the substrate enters the active site, binding of penicillin to the reactive iron species via the sulfur group subsequently expels succinate and leads to oxidative ring expansion of the substrate
-
-
-
additional information
?
-
substrate specificity, oxacillin is no substrate
-
-
-
additional information
?
-
-
substrate specificity, oxacillin is no substrate
-
-
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(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
P18548
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
P18548
-
-
-
?
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
P18548
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
P18548
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
penicillin mX + 2-oxoglutarate + O2
7-[(3-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
P18548
-
-
-
?
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
Q9P4T5
-
-
-
ir
deacetoxycephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2
P11935
-
-
-
ir
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-adipylaminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
-
?
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
product is the precursor of 7-aminodeacetoxycephalosporanic acid, which is used in industrial applications
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
Q9P4T5
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P11935
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P11935
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P11935
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
ring expansion of penicillin N to a six member cephem ring, the natural enzyme is only able to use 2-oxoglutarate but not 2-oxobutyrate, 3-oxoadipate, 2-oxohexanoic acid or 2-oxo-4-methylpentanoic acid as cosubstrates
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P18548
the enzyme catalyzes the committed step in the biosynthesis of cephalosporin antibiotics
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
committed step in biosynthesis of cephamycin C
-
-
-
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme is involved in catalyzing the biosynthesis of cephalosporins and cephamycin
-
-
-
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
first step in cephamycin C biosynthetic pathway
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring
-
-
ir
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
P18548
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
Q93FD4
first step in cephamycin C biosynthetic pathway
-
-
ir
additional information
?
-
-
the enzyme catalyzes the first and second step in the cephamycin C biosynthetic pathway
-
-
-
additional information
?
-
Q9P4T5
the enzyme catalyzes the first and second step in the cephamycin C biosynthetic pathway
-
-
-
additional information
?
-
-
a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
P18548
enzyme is able to convert a wide range of penicillin substrates differing in their side chains, it is a member of 2-oxoglutarate-dependent dioxygenase protein family, the mechanism involves initial activation of the iron centre by 2-oxoglutarate binding followed by oxidative decarboxylation of 2-oxoglutarate to generate oxidizing ferryl species with succinate and carbon dioxide as by-products, succinate remains bound and stabilizes the reactive iron species until the substrate enters the active site, binding of penicillin to the reactive iron species via the sulfur group subsequently expels succinate and leads to oxidative ring expansion of the substrate
-
-
-
additional information
?
-
-
Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G
-
-
-
additional information
?
-
-
Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G
-
-
-
additional information
?
-
-
does not use isopenicillin N or 6-aminopenicillanic acid as substrates, the natural enzyme is only able to use 2-oxoglutarate but not 2-ketobutyrate, 3-ketoadipate, 2-ketohexanoic acid, or 2-keto-4-methylpentanoic acid as cosubstrates
-
-
-
additional information
?
-
P18548
enzyme is able to convert a wide range of penicillin substrates differing in their side chains, it is a member of 2-oxoglutarate-dependent dioxygenase protein family, the mechanism involves initial activation of the iron centre by 2-oxoglutarate binding followed by oxidative decarboxylation of 2-oxoglutarate to generate oxidizing ferryl species with succinate and carbon dioxide as by-products, succinate remains bound and stabilizes the reactive iron species until the substrate enters the active site, binding of penicillin to the reactive iron species via the sulfur group subsequently expels succinate and leads to oxidative ring expansion of the substrate
-
-
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Fe3+
Iron
additional information
Fe2+
dependent on, iron cofactor is bound by D184, H186, and H244
Fe2+
-
required, cannot be substituted by other divalent metal ions
Fe2+
-
bound to the active site, forms a reactive iron-oxygen reaction intermediate during catalysis, complex structure in native and His-tagged enzyme
Fe2+
acts as cofactor, the iron-binding centre in this family of enzymes essentially provides versatility and flexibility in catalysing a variety of oxidative reactions that include desaturation, ring expansion, and hydroxylation
Fe2+
-
required, cannot be substituted by other divalent metal ions
Fe2+
required, residues M180 and I305 are involved in Fe2+-binding
additional information
-
Fe2+ cannot be replaced by Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Ni2+ or Zn2+
additional information
-
Fe2+ cannot be replaced by Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Ni2+ or Zn2+
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
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
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
ampicillin
-
; 2.8 mM, 13% inhibition of the formation of deacetoxycephalosporin C
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
-
5,5'-dithiobis-2-nitrobenzoic acid
-
1 mM, 100% inhibition of expandase reaction, EC 1.14.20.1
5,5'-dithiobis-2-nitrobenzoic acid
-
0.01 mM, 30% inhibition, reactivation by dithiothreitol
iodoacetic acid
-
1 mM, 4% inhibition of expandase reaction, EC 1.14.20.1
N-ethylmaleimide
-
1 mM, 51% inhibition of expandase reaction, EC 1.14.20.1
o-phenanthroline
-
0.6 mM, 100% inhibition of expandase reaction, EC 1.14.20.1
p-hydroxymercuribenzoate
-
1 mM, 100% inhibition of expandase reaction, EC 1.14.20.1
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
dithiothreitol
-
; stimulates activity of native and recombinant enzyme
additional information
-
best activity is obtained by adding to the enzyme a mixture of ATP, Fe2+, and ascorbate, followed by addition of 2-oxoglutarate, and then penicillin N. Simultaneous addition of ATP, Fe2+, and ascorbate is preferable to sequential addition
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.38
ampicillin
-
mutant C281Y/N304A, pH 7.4, 30°C; mutant V275I7N304K, pH 7.4, 30°C
0.79
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F
1.3
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L
1.9
ampicillin
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F
2.6
ampicillin
-
pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme; pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L; wild-type enzyme
3.3
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q
4.1
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H
4.2
ampicillin
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H and R258A
4.5
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A
6.6
ampicillin
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q
13
ampicillin
-
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
15
ampicillin
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K
24
ampicillin
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
0.014
penicillin G
recombinant clone FF8 derived from gene shuffling
0.19
penicillin G
mutant C155Y/Y184H/V275I/C281Y; mutant enzyme C155Y/Y184H/V275I/C281Y
0.25
penicillin G
mutant enzyme V275I/I305M; mutant V275I/I305M
0.75
penicillin G
-
pH 6.5, 30°C, recombinant mutants G79E and I305M
0.75
penicillin G
mutant enzyme G79E; mutant enzyme I305M; mutant G79E; mutant I305M
0.79
penicillin G
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258Q
0.84
penicillin G
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F
1.1
penicillin G
-
pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme; wild-type enzyme
1.2
penicillin G
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L
1.5
penicillin G
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258H and R258Q
1.6
penicillin G
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F
1.6
penicillin G
-
mutant C155Y/Y184H/V275I/C281Y, pH and temperature not specified in the publication
1.62
penicillin G
-
mutant I305M/S261M, pH and temperature not specified in the publication
1.7
penicillin G
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L
1.8
penicillin G
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A
1.9
penicillin G
-
wild-type enzyme, pH not specified in the publication, temperature not specified in the publication
1.92
penicillin G
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
2
penicillin G
30°C, triple mutant with truncation at residue 310, M306I and N305L; pH 7.5, 30°C, recombinant mutant DELTA310/N305L/M306I
2.1
penicillin G
-
mutant I305L, pH and temperature not specified in the publication
2.49
penicillin G
-
mutant I305M/T213V/S261M, pH and temperature not specified in the publication
3
penicillin G
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H
3.22
penicillin G
-
mutant I305M/T213V, pH and temperature not specified in the publication
3.58
penicillin G
-
mutant I305M, pH and temperature not specified in the publication
4.7
penicillin G
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
4.71
penicillin G
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
5.28
penicillin G
-
recombinant mutant S261A, pH not specified in the publication, 30°C
6.4
penicillin G
-
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
6.42
penicillin G
30°C, double mutant with truncation at residue 310 and M306I
7
penicillin G
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K
8.2
penicillin G
-
mutant R308I, pH not specified in the publication, temperature not specified in the publication
9.9
penicillin G
-
recombinant mutant T213V, pH not specified in the publication, 30°C
11
penicillin G
-
mutant R308L, pH not specified in the publication, temperature not specified in the publication
14.38
penicillin G
-
wild-type enzyme, pH and temperature not specified in the publication
14.38
penicillin G
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
41.16
penicillin G
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
48.1
penicillin G
-
recombinant mutant S261M, pH not specified in the publication, 30°C
0.006
Penicillin N
-
pH 6.5, 30°C, recombinant mutants C281Y and I305L
0.012
Penicillin N
-
pH 6.5, 30°C, recombinant mutants V275I and I305M
additional information
additional information
-
KM-values for mutant enzymes with penicillin G or Ampicillin as prime substrate and 2-oxo-4-methylpentanoate, 2-oxoglutarate or 2-oxohexanoate as cosubstrate
-
additional information
additional information
-
Km-values for mutant enzymes with penicillin N and penicillin G as substrate
-
additional information
additional information
-
KM-value of Fe2+ is 0.071 mM
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.005
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H
0.007
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258H
0.008
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258A
0.009
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L; pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
0.011
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258A and R258F; pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
0.012
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258L and R258F
0.013
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K
0.014
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q; pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme; wild-type enzyme
0.017
ampicillin
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q
0.037
ampicillin
Streptomyces clavuligerus
-
mutant C281Y/R306L, pH 7.4, 30°C; mutant V275I/R306L, pH 7.4, 30°C
0.04
ampicillin
Streptomyces clavuligerus
-
mutant N304A, pH 7.4, 30°C; mutant V275I7N304K, pH 7.4, 30°C
0.042
ampicillin
Streptomyces clavuligerus
-
mutant C281F, pH 7.4, 30°C; mutant V275I/N304A, pH 7.4, 30°C
0.043
ampicillin
Streptomyces clavuligerus
-
mutant C281Y7N304R, pH 7.4, 30°C; mutant C281Y/N304L, pH 7.4, 30°C; mutant V275I/N304L, pH 7.4, 30°C
0.044
ampicillin
Streptomyces clavuligerus
-
mutant C281Y, pH 7.4, 30°C; mutant V275I, pH 7.4, 30°C
0.046
ampicillin
Streptomyces clavuligerus
-
mutant C281Y/N304K, pH 7.4, 30°C; mutant C281Y/R307L, pH 7.4, 30°C
0.05
ampicillin
Streptomyces clavuligerus
-
mutant R306L, pH 7.4, 30°C; mutant R307L, pH 7.4, 30°C
0.052
ampicillin
Streptomyces clavuligerus
-
mutant C281Y/N304A, pH 7.4, 30°C; mutant N304R, pH 7.4, 30°C
0.001
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
0.0057
penicillin G
Acremonium chrysogenum
-
wild-type enzyme, pH not specified in the publication, temperature not specified in the publication
0.01
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K; pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q
0.013
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258H
0.014
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H
0.019
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L
0.02
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258K, R258A, and R258L
0.021
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q; pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme; wild-type enzyme
0.024
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F
0.025
penicillin G
Streptomyces clavuligerus
-
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F
0.0297
penicillin G
Streptomyces clavuligerus
P18548
recombinant FF2 derived from gene shuffling
0.048
penicillin G
Acremonium chrysogenum
-
mutant R308L, pH not specified in the publication, temperature not specified in the publication
0.054
penicillin G
Streptomyces clavuligerus
-
wild-type enzyme, pH and temperature not specified in the publication
0.054
penicillin G
Streptomyces clavuligerus
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
0.057
penicillin G
Streptomyces clavuligerus
-
recombinant mutant T213V, pH not specified in the publication, 30°C
0.064
penicillin G
Streptomyces clavuligerus
-
recombinant mutant S261A, pH not specified in the publication, 30°C
0.069
penicillin G
Acremonium chrysogenum
-
mutant R308I, pH not specified in the publication, temperature not specified in the publication
0.07
penicillin G
Acremonium chrysogenum
P11935
30°C, triple mutant with truncation at residue 310, M306I and N305L
0.073
penicillin G
Acremonium chrysogenum
P11935
30°C, wild-type; pH 7.5, 30°C, recombinant wild-type enzyme
0.081
penicillin G
Streptomyces clavuligerus
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
0.091
penicillin G
Acremonium chrysogenum
P11935
30°C, double mutant with truncation at residue 310 and M306I
0.1398
penicillin G
Streptomyces clavuligerus
P18548
mutant C155Y/Y184H/V275I/C281Y; mutant enzyme C155Y/Y184H/V275I/C281Y
0.1458
penicillin G
Streptomyces clavuligerus
P18548
mutant enzyme V275I/I305M; mutant V275I/I305 M
0.225
penicillin G
Streptomyces clavuligerus
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
0.23
penicillin G
Streptomyces clavuligerus
-
mutant C155Y/Y184H/V275I/C281Y, pH and temperature not specified in the publication
0.37
penicillin G
Streptomyces clavuligerus
-
mutant I305L, pH and temperature not specified in the publication
0.433
penicillin G
Streptomyces clavuligerus
-
recombinant mutant S261M, pH not specified in the publication, 30°C
0.537
penicillin G
Streptomyces clavuligerus
-
mutant I305M/S261M, pH and temperature not specified in the publication
0.589
penicillin G
Streptomyces clavuligerus
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
0.65
penicillin G
Streptomyces clavuligerus
-
mutant I305M, pH and temperature not specified in the publication
0.654
penicillin G
Streptomyces clavuligerus
-
mutant I305M/T213V, pH and temperature not specified in the publication
0.668
penicillin G
Streptomyces clavuligerus
-
mutant I305M/T213V/S261M, pH and temperature not specified in the publication
0.02
Penicillin N
Streptomyces clavuligerus
-
mutant enzyme DELTAI305-310, DELTAR306-310 and mutant DELTAR307-310
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.003
penicillin G
Acremonium chrysogenum
-
wild-type enzyme, pH not specified in the publication, temperature not specified in the publication
181
0.0038
penicillin G
Streptomyces clavuligerus
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
181
0.004
penicillin G
Streptomyces clavuligerus
-
wild-type enzyme, pH and temperature not specified in the publication
181
0.0045
penicillin G
Acremonium chrysogenum
-
mutant R308L, pH not specified in the publication, temperature not specified in the publication
181
0.0055
penicillin G
Streptomyces clavuligerus
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
181
0.0058
penicillin G
Streptomyces clavuligerus
-
recombinant mutant T213V, pH not specified in the publication, 30°C
181
0.0085
penicillin G
Acremonium chrysogenum
-
mutant R308I, pH not specified in the publication, temperature not specified in the publication
181
0.009
penicillin G
Streptomyces clavuligerus
-
recombinant mutant S261M, pH not specified in the publication, 30°C
181
0.0119
penicillin G
Streptomyces clavuligerus
-
recombinant mutant S261A, pH not specified in the publication, 30°C
181
0.0171
penicillin G
Streptomyces clavuligerus
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
181
0.144
penicillin G
Streptomyces clavuligerus
-
mutant C155Y/Y184H/V275I/C281Y, pH and temperature not specified in the publication
181
0.176
penicillin G
Streptomyces clavuligerus
-
mutant I305L, pH and temperature not specified in the publication
181
0.182
penicillin G
Streptomyces clavuligerus
-
mutant I305M, pH and temperature not specified in the publication
181
0.203
penicillin G
Streptomyces clavuligerus
-
mutant I305M/T213V, pH and temperature not specified in the publication
181
0.268
penicillin G
Streptomyces clavuligerus
-
mutant I305M/T213V/S261M, pH and temperature not specified in the publication
181
0.307
penicillin G
Streptomyces clavuligerus
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
181
0.332
penicillin G
Streptomyces clavuligerus
-
mutant I305M/S261M, pH and temperature not specified in the publication
181
0.736
penicillin G
Streptomyces clavuligerus
P18548
mutant enzyme C155Y/Y184H/V275I/C281Y
181
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.014
purified recombinant wild-type and truncated mutant enzyme, substrate metampicillin
0.023
purified recombinant truncated mutant enzyme, substrate carbenicillin
0.026
0.039
purified recombinant wild-type and truncated mutant enzyme, substrate ampicillin, determined by bioassay
0.071
purified recombinant truncated mutant enzyme, substrate phenethicillin
0.074
purified recombinant wild-type enzyme, substrate phenethicillin
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
0.178
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
0.458
recombinant enzyme, substrate ampicillin, determined by bioassay
0.932
recombinant enzyme, substrate ampicillin; recombinant enzyme, substrate ampicillin, determined by HPLC
2.221
-
recombinant enzyme, substrate ampicillin, determined by HPLC
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
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
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
additional information
0.026
purified recombinant wild-type enzyme, substrate carbenicillin
additional information
activities of wild-type enzyme in ring-expansion and hydroxylation, cosubstrate and substrate conversion rate
additional information
-
relative activities of wild-type and R308 mutants with penicillin variants and analogue substrates, overview
additional information
-
activity with different substrates and assay methods of recombinant wild-type and mutant enzymes, overview
additional information
-
activity of recombinant wild-type and mutant enzymes with substrate penicillin G and penicillin N
additional information
-
160% relative activity with 10 mM substrate penicillin G compared to the activity detected with 1 mM penicillin G
additional information
quaternary mutant (C155Y/Y184H/V275I/C281Y) with enhanced activity and without substrate inhibition; the activity of scDAOCS very much depends on the stability of the enzyme maintained by equilibrium in a pool of monomeric and trimeric forms of scDAOCS which can be disrupted by introduction of a relatively small His-tag at the N-terminus of the enzyme; to achieve optimum conditions for the conversion of penicillin to a cephalosporin, a reaction mixture containing 50 mM Tris-HCl (pH 7.4), 10 mM KCl, 10 mM MgSO4, 0.6 mM ascorbic acid, 0.8 mM ATP, 0.04 mM FeSO4, 0.6 mM 2-oxoglutarate, and 0.28 mM penicillin N is used for detection of the ring-expansion activity of DAOCS from Acremonium chrysogenum, DAOCS is unable to convert penicillin G and other penicillin N analogs under this optimum conditions described for penicillin N
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
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
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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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)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
28900
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
36000
-
x * 36000, SDS-PAGE; x * 36000, SDS-PAGE, x * 34519, calculated
36348
x * 36348, wild-type enzyme, electrospray mass spectrometry
40000
60000
62000
x * 60000, recombinant truncated mutant enzyme, SDS-PAGE, x * 62000, recombinant wild-type enzyme
60000
x * 60000, recombinant truncated mutant enzyme, SDS-PAGE, x * 62000, recombinant wild-type enzyme
60000
-
x * 60000, recombinant enzyme, SDS-PAGE; x * 60000, SDS-PAGE
60000
x * 60000, recombinant enzyme, SDS-PAGE; x * 60000, SDS-PAGE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
trimer
additional information
?
x * 60000, recombinant truncated mutant enzyme, SDS-PAGE, x * 62000, recombinant wild-type enzyme
?
-
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE
?
-
x * 34554, in solution the monomeric apoenzyme is in equilibrium with atrimeric form, electrospray mass spectrometry
?
-
x * 34519, calculation from nucleotide sequence; x * 36000, SDS-PAGE; x * 36000, SDS-PAGE, x * 34519, calculated
?
x * 60000, recombinant enzyme, SDS-PAGE; x * 60000, SDS-PAGE
monomer
-
DAOC synthase, recombinant DAOCS of Streptomyces clavuligerus is a monomer but is in equilibrium with a trimeric form of 92900 Da
monomer
upon addition of Fe(II) and 2-oxoglutarate, scDAOCS dissociates into monomers, determined by gelfiltration and dynamic laser light scattering studies which reveal the existence of an equilibrium between the monomeric and trimeric scDAOCS in the absence of Fe2+
monomer
-
upon addition of Fe(II) and 2-oxoglutarate, scDAOCS dissociates into monomers, determined by gelfiltration and dynamic laser light scattering studies which reveal the existence of an equilibrium between the monomeric and trimeric scDAOCS in the absence of Fe2+
-
trimer
native state, the C-terminus of one molecule (residues 308-311) penetrates the active site of its neighbouring molecule in a cyclical fashion
trimer
-
3 * 30966, DAOCS is in equilibrium with a trimeric form which is the form that crystallizes
trimer
-
3 * 30966, DAOCS is in equilibrium with a trimeric form which is the form that crystallizes
-
trimer
-
native state, the C-terminus of one molecule (residues 308-311) penetrates the active site of its neighbouring molecule in a cyclical fashion
-
additional information
MW of mutant enzyme determined by electrospray mass spectrometry; structural model of enzyme
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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
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
increasing ionic strength to 100 decreases stability of the enzyme
-
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
-
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
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
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
-
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
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
recombinant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
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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 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
-
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 as GST-fusion protein in Escherichia coli strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
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
N305L
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
R308L
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
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
C155Y/Y184H/V275I/C281Y
C281F
-
substrate ampicillin, 191%, penicillin G, 264%, phenethicillin, 300%, and carbenicillin, 518% of wild-type activity
C281Y
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
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
G79E
H244Q
I305L
I305M
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
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M/T213V/S261M
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
L277Q
L277Q/I305M
-
610% relative activity with 1 mM and 520% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M188I
-
90% relative activity with 1 mM and 190% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, epPCR random mutagenesis
M188V
M188V/V275I/G300V
-
440% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T
M73T/C281Y
-
610% relative activity with 1 mM and 530% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/G300V/I305L
-
640% relative activity with 1 mM and 380% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/I118V/A140V/H244Q/C281Y
-
450% relative activity with 1 mM and 350% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
M73T/I277Q
-
350% relative activity with 1 mM and 350% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
N304A
N304A/R306L
-
substrate ampicillin, 214%, penicillin G, 114%, phenethicillin, 319%, and carbenicillin, 393% of wild-type activity
N304K
N304K/R306L
-
substrate ampicillin, 245%, penicillin G, 80%, phenethicillin, 380%, and carbenicillin, 482% of wild-type activity
N304L
N304L/R306L
-
substrate ampicillin, 145%, penicillin G, 40%, phenethicillin, 185%, and carbenicillin, 218% of wild-type activity
N304M
-
substrate ampicillin, 120%, penicillin G,92%, phenethicillin, 127%, and carbenicillin, 161% of wild-type activity
N304M/R306L
-
substrate ampicillin, 194%, penicillin G, 72%, phenethicillin, 245%, and carbenicillin, 232% of wild-type activity
N304R
N304R/R306L
-
substrate ampicillin, 256%, penicillin G, 92%, phenethicillin, 344%, and carbenicillin, 514% of wild-type activity
Q126M
R135Q/C155Y/R179Q/M188V/I305M
-
360% relative activity with 1 mM and 290% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
R160L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
R258A
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme; wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme
R258F
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme; wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme
R258H
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme; wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme
R258K
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme
R258L
R258Q
R266L
R266Q
-
2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present
R306A
-
substrate ampicillin, 46%, penicillin G, 37%, phenethicillin, 30%, and carbenicillin, 27% of wild-type activity
R306C
-
substrate ampicillin, 18%, penicillin G, 13%, phenethicillin, 11%, of wild-type activity
R306D
-
substrate ampicillin, 8%, penicillin G, 4%, phenethicillin, 4%, of wild-type activity
R306E
-
substrate ampicillin, 8%, penicillin G, 7%, phenethicillin, 6%,of wild-type activity
R306F
-
substrate ampicillin, 55%, penicillin G, 41%, phenethicillin, 44%, and carbenicillin, 39% of wild-type activity
R306G
-
substrate ampicillin, 21%, penicillin G, 27%, phenethicillin, 19%, of wild-type activity
R306H
-
substrate ampicillin, 16%, penicillin G, 10%, phenethicillin, 8%, of wild-type activity
R306I
R306K
-
substrate ampicillin, 50%, penicillin G, 53%, phenethicillin, 45%, and carbenicillin, 32% of wild-type activity
R306L
R306M
R306N
-
substrate ampicillin, 16%, penicillin G, 14%, phenethicillin, 12%, and carbenicillin, 16% of wild-type activity
R306P
R306Q
-
substrate ampicillin, 62%, penicillin G, 67%, phenethicillin, 54%, and carbenicillin, 62% of wild-type activity
R306S
-
substrate ampicillin, 39%, penicillin G, 37%, phenethicillin, 27%, and carbenicillin, 30% of wild-type activity
R306T
-
substrate ampicillin, 40%, penicillin G, 52%, phenethicillin, 32%, and carbenicillin, 34% of wild-type activity
R306V
-
substrate ampicillin, 34%, penicillin G, 28%, phenethicillin, 36%, and carbenicillin, 32% of wild-type activity
R306W
-
substrate ampicillin, 90%, penicillin G, 61%, phenethicillin, 82%, and carbenicillin, 83% of wild-type activity
R306Y
-
substrate ampicillin, 70%, penicillin G, 59%, phenethicillin, 61%, and carbenicillin, 60% of wild-type activity
R307L
R307Q
-
mutation enhances penicillin N conversion compared to the level of wild-type enzyme, turnover number for penicillin N is increased, no enhancement in activity with penicillin G as substrate. Mutation increase the Km-value by 10fold, but has little effect on the turnover number for penicillin G
R74I
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme
R74L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
R74Q
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N and penicillin G compared to wild type enzyme. Penicillin oxidation is reduced relative to the wild type enzyme
R74Q/R266I
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R74Q/R266Q
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R75I/D270G
-
2-oxoglutarate conversion is not stimulated by penicillin substrates
R75Q
-
turnover number and Km-values are similar to that for wild-type enzyme. 2-Oxoglutarate conversion is significantly stimulated in presence of penicillin N compared to wild type enzyme
S261A
S261I
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S261L
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S261M
S261V
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S59T
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
T213V
T42A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
T91A
V275I
V275I/C281Y
-
260% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/C281Y/G300V
-
620% relative activity with 1 mM and 380% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/C281Y/I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, over 32fold increased activity compared to the wild-type enzyme
V275I/I305L
-
310% relative activity with 1 mM and 330% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
V275I/I305M
V275I/N304A
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substrate ampicillin, 265%, penicillin G, 233%, phenethicillin, 330%, and carbenicillin,721% of wild-type activity
V275I/N304K
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substrate ampicillin, 334%, penicillin G, 294%, phenethicillin, 635%, and carbenicillin, 867% of wild-type activity
V275I/N304L
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substrate ampicillin, 225%, penicillin G, 141%, phenethicillin, 245%, and carbenicillin, 524% of wild-type activity
V275I/N304M
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substrate ampicillin, 160%, penicillin G, 174%, phenethicillin, 261%, and carbenicillin, 230% of wild-type activity
V275I/N304R
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substrate ampicillin, 360%, penicillin G, 381%, phenethicillin, 698%, and carbenicillin,814% of wild-type activity
V275I/R306L
-
substrate ampicillin, 343%, penicillin G, 168%, phenethicillin, 379%, and carbenicillin, 665% of wild-type activity
V275I/R307L
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substrate ampicillin, 172%, penicillin G, 169%, phenethicillin, 238%, and carbenicillin, 333% of wild-type activity
V275L
-
substrate ampicillin, 85%, penicillin G, 130%, phenethicillin, 99%, and carbenicillin, 113% of wild-type activity
V51A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184A
-
site-directed mutagenesis, the mutant shows activity increased to 143.9% with penicillin G compared to the wild-type
Y184H
Y184H/H244Q/T259I/L277Q
-
320% relative activity with 1 mM and 310% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
Y184H/M188I/C281Y/I305L
-
610% relative activity with 1 mM and 460% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
Y184I
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184L
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
Y184M
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
C37S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
T42A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
V51A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
-
Y184A
-
site-directed mutagenesis, the mutant shows activity increased to 143.9% with penicillin G compared to the wild-type
-
R258L
-
mutant have the same catalytic activity as the R258Q mutant when 2-oxo-4-methylpentanoate is used as a co-substrate
-
R258Q
-
mutation in scDAOCS almost abolishes the oxidation of both penicillin N and penicillin G, aliphatic 2-oxoacids (2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate), which can not replace the function of 2-oxoglutarate for the wild-type enzyme, are able to rescue the catalytic activity of the R258Q mutant to the level observed for the wild-type enzyme
-
R266L
-
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
-
R74L
-
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
-
additional information
N305L
107% of wild-type ring expansion activity, 85% of wild-type hydroxylation activity
N305L
-
improved ability to convert penicillin analogs in ring expansion reaction of EC 1.14.20.1
R308L
-
improved ability to convert penicillin analogs in ring expansion reaction of EC 1.14.20.1
R308L
-
site-directed mutagenesis, the mutant shows significant improvement in the ability to convert penicillin analogues compared to the wild-type enzyme
C155Y
-
90% relative activity with 1 mM and 180% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, 1.5fold reduction in Km, penicillin G as substrate
C155Y
the mutant has 1.5fold increment in kcat/Km value when compared with the wild type enzyme
C155Y/Y184H/V275I/C281Y
-
580% relative activity with 1 mM and 670% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
C155Y/Y184H/V275I/C281Y
mutant with enhanced activity and without substrate inhibition
C155Y/Y184H/V275I/C281Y
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
C281Y
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to the wild-type enzyme
C281Y
-
substrate ampicillin, 166%, penicillin G, 251%, phenethicillin, 280%, and carbenicillin, 572% of wild-type activity
C281Y
the mutant has 6.1fold increment in kcat/Km value when compared with the wild type enzyme
C281Y/I305M
-
substrate ampicillin, 491%, penicillin G, 495%, phenethicillin, 1109%, and carbenicillin, 1347% of wild-type activity
C281Y/I305M
kcat/Km values of double-mutant scDAOCSs are higher than that of the wild-type enzyme for ampicillin conversion, improvement in the enzyme’s catalytic effciency (68fold increment)
C281Y/N304R
-
substrate ampicillin, 430%, penicillin G, 441%, phenethicillin, 1041%, and carbenicillin, 1309% of wild-type activity
C281Y/N304R
kcat/Km values of double-mutant scDAOCSs are higher than that of the wild-type enzyme for ampicillin conversion, improvement in the enzyme’s catalytic effciency (101fold increment)
G79E
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to the wild-type enzyme
G79E
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 2.3fold increment in kcat/Km value when compared with the wild type enzyme
H244Q
-
140% relative activity with 1 mM and 270% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, 1.9fold reduction in Km, penicillin G as substrate, epPCR random mutagenesis
H244Q
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 2.8fold increment in kcat/Km value when compared with the wild type enzyme
I305L
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 6-14fold increased activity compared to the wild-type enzyme
I305L
mutation is able to increase the activity of the enzyme; the mutant has 6.4fold increment in kcat/Km value when compared with the wild type enzyme
I305L
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 6-14fold increased activity compared to the wild-type enzyme
I305M
-
substrate ampicillin, 313%, penicillin G, 234%, phenethicillin, 318%, and carbenicillin, 450% of wild-type activity
I305M
increase of the turnover rate for ampicillin conversion; mutation is able to increase the activity of the enzyme; the mutant has 10.8fold increment in kcat/Km value when compared with the wild type enzyme
I305M
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
L277Q
-
270% relative activity with 1 mM and 410% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, 2.5fold reduction in Km, penicillin G as substrate
L277Q
mutant has the highest catalytic effciency for penicillin G (6fold increment); the mutant has 5.7fold increment in kcat/Km value when compared with the wild type enzyme
M188V
-
150% relative activity with 1 mM and 190% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, epPCR random mutagenesis
M188V
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 3.3fold increment in kcat/Km value when compared with the wild type enzyme
M73T
-
180% relative activity with 1 mM and 250% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G, 3.5fold reduction in Km for penicillin N and 2.3fold reduction in Km for penicillin G, epPCR random mutagenesis
M73T
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 4.7fold increment in kcat/Km value when compared with the wild type enzyme
N304A
-
substrate ampicillin, 192%, penicillin G, 163%, phenethicillin, 163%, and carbenicillin, 242% of wild-type activity
N304A
mutation is able to increase the activity of the enzyme
N304K
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 6-14fold increased activity compared to the wild-type enzyme
N304K
-
substrate ampicillin, 270%, penicillin G, 237%, phenethicillin, 211%, an carbenicillin, 404% of wild-type activity
N304K
improvement of the substrate-binding affinity of the enzyme for ampicillin conversion; mutation is able to increase the activity of the enzyme; the mutant has 14.2fold increment in kcat/Km value when compared with the wild type enzyme
N304L
-
mutant enzyme with improved efficiency in penicillin conversion
N304L
-
substrate ampicillin, 163%, penicillin G, 129%, phenethicillin, 173%, and carbenicillin, 235% of wild-type activity
N304R
-
substrate ampicillin, 346%, penicillin G, 263%, phenethicillin, 360%, and carbenicillin, 398% of wild-type activity
N304R
improvement of the substrate-binding affinity of the enzyme for ampicillin conversion
Q126M
-
site-directed mutagenesis, the mutant shows activity increased to 281.5% with penicillin G compared to the wild-type
R258L
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme; wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme
R258L
mutant have the same catalytic activity as the R258Q mutant when 2-oxo-4-methylpentanoate is used as a co-substrate
R258Q
-
site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme
R258Q
mutation in scDAOCS almost abolishes the oxidation of both penicillin N and penicillin G, aliphatic 2-oxoacids (2-oxo-4-methylpentanoate and 2-oxo-3-methylbutanoate), which can not replace the function of 2-oxoglutarate for the wild-type enzyme, are able to rescue the catalytic activity of the R258Q mutant to the level observed for the wild-type enzyme
R266L
-
2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present
R266L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
R306I
-
substrate ampicillin, 168%, penicillin G, 113%, phenethicillin, 149%, and carbenicillin, 167% of wild-type activity
R306I
replacement of R306 with amino acids structurally similar to leucine is able to improve the enzyme activity via hydrophobic interaction with the surrounding residues
R306L
-
mutation enhances penicillin N conversion compared to the level of wild-type enzyme, turnover number for penicillin N is increased, no enhancement in activity with penicillin G as substrate, little effect on kinetic values using penicillin G as substrate
R306L
-
mutant enzyme with improved efficiency in penicillin conversion
R306L
-
substrate ampicillin, 186%, penicillin G, 105%, phenethicillin, 173%, and carbenicillin, 283% of wild-type activity
R306M
-
substrate ampicillin, 113%, penicillin G, 102%, phenethicillin, 115%, and carbenicillin, 122% of wild-type activity
R306M
replacement of R306 with amino acids structurally similar to leucine is able to improve the enzyme activity via hydrophobic interaction with the surrounding residues
R307L
-
mutant enzyme with improved efficiency in penicillin conversion
R307L
-
substrate ampicillin, 133%, penicillin G, 124%, phenethicillin, 122%, and carbenicillin, 108% of wild-type activity
S261A
-
site-directed mutagenesis, the mutant shows activity increased to 128.8% with penicillin G compared to the wild-type
S261M
-
site-directed mutagenesis, the mutant shows activity increased to 158.4% with penicillin G compared to the wild-type
T213V
-
site-directed mutagenesis, the mutant shows activity increased to 172.4% with penicillin G compared to the wild-type
T91A
-
110% 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, 1.9fold reduction in Km, penicillin G as substrate, epPCR random mutagenesis
T91A
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 2.1fold increment in kcat/Km value when compared with the wild type enzyme
V275I
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to the wild-type enzyme
V275I
-
substrate ampicillin, 129%, penicillin G, 157%, phenethicillin, 150%, and carbenicillin, 227% of wild-type activity
V275I
the mutant has 1.7fold increment in kcat/Km value when compared with the wild type enzyme
V275I/I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 32fold increased activity compared to the wild-type enzyme
V275I/I305M
-
substrate ampicillin, 406%, penicillin G, 420%, phenethicillin, 685%, and carbenicillin, 1057% of wild-type activity
V275I/I305M
the mutant has 32.4fold increment in kcat/Km value when compared with the wild type enzyme
Y184H
-
200% relative activity with 1 mM and 330% relative activity with 10 mM substrate penicillin G compared to wild-type activity detected with 1 mM penicillin G
Y184H
mutant has approximately 2 to 5fold increment in kcat/Km values when compared with the wild-type enzyme; the mutant has 4.7fold increment in kcat/Km value when compared with the wild type enzyme
additional information
DELTA310 is a C-terminally truncated mutant lacking residues 1-309, the mutant shows 2fold enhanced ring expansion activity with the artificial substrate penicillin G; truncation of C-terminus to residue 310, 2fold enhancement of ring expansion reaction of penicillin G. Double mutant with truncation at residue 310 and M306I, selective catalyzation of ring expansion. Triple mutant with truncation at residue 310, M306I and N305L, selective catalization of ring expansion with improved kinetic parameters
additional information
-
mutations to obtain a wider substrate specificity for DAOCS are very important since modification of the DAOCS activity to accept hydrophobic penicillins is of great industrial interest
additional information
-
the truncation of the C-terminus at position 310 in the wild-type enzyme results in reduction of indiscriminate conversion of penicillin analog but this defect is compensated by the replacement of asparagine with leucine at position 304
additional information
-
shortening of the C-terminus by more than 4 residues reduces enzyme activity and alters the crystal structure from merohedrally twinned trimeric crystals to a non-trimeric structure with a free C-terminal arm, crystals show no twinning
additional information
-
since the natural substrate of the enzyme, penicillin N, is not available in large quantities, engineering of a scDAOCS capable of converting the cheap and easily available substrate penicillin G is valuable for large-scale production of 7-aminodeacetoxycephalosporanic acid, 7-ADCA
additional information
-
since the natural substrate of the enzyme, penicillin N, is not available in large quantities, engineering of a scDAOCS capable of converting the cheap and easily available substrate penicillin G is valuable for large-scale production of 7-aminodeacetoxycephalosporanic acid, 7-ADCA. Ten sites, Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213, are mutated to 21 point mutants
additional information
-
since the natural substrate of the enzyme, penicillin N, is not available in large quantities, engineering of a scDAOCS capable of converting the cheap and easily available substrate penicillin G is valuable for large-scale production of 7-aminodeacetoxycephalosporanic acid, 7-ADCA. Ten sites, Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213, are mutated to 21 point mutants
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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
LITERATURE
medicine
synthesis
synthesis
-
enzyme produces the 7-aminodeacetoxycephalosporanic acid precursor utilized in industrial applications, optimization of enzyme activity for activity with the substrate penicillin G
synthesis
-
expression in Escherichia coli, overexpression as an insoluble and inactive enzyme, elaboration of refolding scheme resulting in highliy active and moderately stable enzyme
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LINKS TO OTHER DATABASES (specific for EC-Number 1.14.20.1)
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