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Information on EC 1.14.20.1 - deacetoxycephalosporin-C synthase and Organism(s) Streptomyces clavuligerus and UniProt Accession P18548
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1.14.20.1 deacetoxycephalosporin-C synthaseIUBMB Comments
Forms part of the penicillin biosynthesis pathway (for pathway, click here).
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Word Map
- 1.14.20.1
- clavuligerus
- chrysogenum
- acremonium
- isopenicillin
- penicillium
- beta-lactams
-
epimerase
-
cephamycins
- cephalosporium
-
ring-expansion
-
7-aminodeacetoxycephalosporanic
-
pcbab
-
ironii
-
cephem
- lactamdurans
- synthesis
-
7-adca
-
2-oxoglutarate-dependent
- cephalexin
- carbenicillin
-
doacs
- biotechnology
- medicine
The taxonomic range for the selected organisms is: Streptomyces clavuligerus
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Reaction Schemes
Synonyms
daocs, deacetoxycephalosporin c synthase, cefef, expandase, scdaocs, penicillin n expandase, daoc/dacs, deacetoxy/deacetylcephalosporin c synthase, acdaoc/dacs, daoc synthase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DAOCS
-
deacetoxycephalosporin C synthase
-
DAOCS
deacetoxycephalosporin-C synthetase
-
-
-
-
penicillin N expandase
-
-
-
-
DAOCS
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
reaction mechanism
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
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O
ping pong reaction mechanism, analysis, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-
SYSTEMATIC NAME
IUBMB Comments
penicillin-N,2-oxoglutarate:oxygen oxidoreductase (ring-expanding)
Forms part of the penicillin biosynthesis pathway (for pathway, click here).
CAS REGISTRY NUMBER
COMMENTARY
85746-10-7
-
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
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
ampicillin + 2-oxo-4-methyl-pentanoic acid + O2
?
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
ampicillin + 2-oxohexanoic acid + O2
?
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
-
-
-
?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
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
-
-
-
?
N-acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-acetyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-adipyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-butyryldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-decanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-heptanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-hexanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N-valeryldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxo-4-methyl-pentanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + 3-methylbutanoate + CO2 + H2O
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
penicillin G + 2-oxo-4-methylpentanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
-
-
?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanate + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
low catalytic effciency towards penicillin G
-
-
?
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-oxohexanoate + O2
phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ?
-
-
-
?
penicillin G + 2-oxohexanoic acid + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + pentanoate + CO2 + H2O
mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme
-
-
ir
penicillin mX + 2-oxoglutarate + O2
7-[(3-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
3-exomethylenecephalosporin C + 2-oxoglutarate + O2
deacetylcephalosporin C + succinate + CO2 + H2O
-
8% of the activity with cephalosporin N, recombinant enzyme
-
?
amoxicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
ampicillin + 2-oxo-4-methylpentanoate + O2
cephalexin + 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
-
?
ampicillin + 2-oxohexanoate + O2
cephalexin + ?
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
-
-
?
carbenicillin + 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
-
-
-
-
?
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
penicillin G + 2-oxoglutarate + O2
phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O
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
-
-
?
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
poor substrate
-
?
ampicillin + 2-oxoglutarate + O2
? + succinate + CO2
-
-
-
?
carbenicillin + 2-oxoglutarate + O2
? + succinate + CO2
-
-
-
?
penicillin G + 2-oxoglutarate + O2
? + succinate + CO2
-
-
-
?
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-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
-
-
-
-
ir
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
the enzyme catalyzes the committed step in the biosynthesis of cephalosporin antibiotics
-
-
?
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
DAOCS-catalyzed penicillin N ring expansion, NMR structure analysis, overview
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
ring expansion of penicillin N
-
-
?
phenethicillin + 2-oxoglutarate + O2
? + succinate + CO2
-
-
-
?
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
-
-
?
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-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 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
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
-
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
-
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 V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
-
-
-
?
penicillin V + 2-oxoglutarate + O2
? + succinate + CO2 + H2O
-
no activity
-
-
?
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
?
-
-
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
?
-
purified recombinant enzyme DAOCS expressed in Escherichia coli catalyzes the ring expansion of penicillin N but shows no hydroxylation activity for DAOC, EC 1.14.11.26
-
-
?
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
?
-
-
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
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(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
-
-
-
?
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-acetylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-adipylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
butyryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-butyrylaminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
D-carboxymethylcysteinyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
?
-
-
-
?
decanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-decanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
heptanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-heptanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
hexanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
7-hexanoylaminodesacetoxycephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
N-nonanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-nonanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-octanoyl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-octanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
N-valeryl-6-aminopenicillanic acid + 2-oxoglutarate + O2
N7-pentanoyldeacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
?
penicillin F + 2-oxoglutarate + O2
7-((3E)-hex-3-enoyl)aminocephalosporanic acid + succinate + CO2 + H2O
-
-
-
?
penicillin G + 2-oxoglutarate + O2
7-phenylacetylaminocephalosporanic acid + succinate + CO2 + H2O
low catalytic effciency towards penicillin G
-
-
?
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
-
-
-
?
penicillin X + 2-oxoglutarate + O2
7-[(4-hydroxyphenyl)acetyl]aminocephalosporanic 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
-
-
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
-
-
-
ir
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
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
-
-
-
-
?
penicillin N + 2-oxoglutarate + O2
deacetoxycephalosporin C + succinate + CO2 + H2O
-
-
-
-
ir
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
-
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
-
-
?
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
?
-
-
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
?
-
-
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
?
-
-
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
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Fe2+
Iron
additional information
-
Fe2+ cannot be replaced by Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Ni2+ or Zn2+
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, residues M180 and I305 are involved in Fe2+-binding
Fe2+
-
required, cannot be substituted by other divalent metal ions
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
5,5'-dithiobis-2-nitrobenzoic acid
-
0.01 mM, 30% inhibition, reactivation by dithiothreitol
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
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
dithiothreitol
-
stimulates activity of native and recombinant enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
2.6
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L
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
0.79
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
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
1.7
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
3
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H
4.7
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
6.4
penicillin G
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
7
penicillin G
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K
20.6
penicillin G
recombinant His-tagged enzyme, pH 7.5, 30°C
0.75
penicillin G
-
pH 6.5, 30°C, recombinant mutants G79E and I305M
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
1.92
penicillin G
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
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
3.22
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
4.71
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
9.9
penicillin G
-
recombinant mutant T213V, pH not specified in the publication, 30°C
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
pre-steady-state and steady-state kinetics, rapid quench flow and stopped-flow kinetics
-
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 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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.005
ampicillin
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H
0.007
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258H
0.008
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258A
0.009
ampicillin
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L
0.009
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
0.011
ampicillin
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258A and R258F
0.011
ampicillin
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
0.012
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258L and R258F
0.013
ampicillin
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K
0.014
ampicillin
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q
0.014
ampicillin
pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme
0.017
ampicillin
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q
0.001
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K
0.01
penicillin G
pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K
0.01
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q
0.013
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258H
0.014
penicillin G
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H
0.019
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L
0.02
penicillin G
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258K, R258A, and R258L
0.021
penicillin G
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q
0.021
penicillin G
pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme
0.024
penicillin G
pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F
0.025
penicillin G
pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F
0.0297
penicillin G
recombinant FF2 derived from gene shuffling
0.056
penicillin G
recombinant His-tagged enzyme, pH 7.5, 30°C
0.02
Penicillin N
mutant enzyme DELTAI305-310, DELTAR306-310 and mutant DELTAR307-310
0.054
penicillin G
-
wild-type enzyme, pH and temperature not specified in the publication
0.054
penicillin G
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
0.057
penicillin G
-
recombinant mutant T213V, pH not specified in the publication, 30°C
0.064
penicillin G
-
recombinant mutant S261A, pH not specified in the publication, 30°C
0.081
penicillin G
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
0.225
penicillin G
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
0.23
penicillin G
-
mutant C155Y/Y184H/V275I/C281Y, pH and temperature not specified in the publication
0.37
penicillin G
-
mutant I305L, pH and temperature not specified in the publication
0.433
penicillin G
-
recombinant mutant S261M, pH not specified in the publication, 30°C
0.537
penicillin G
-
mutant I305M/S261M, pH and temperature not specified in the publication
0.589
penicillin G
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
0.65
penicillin G
-
mutant I305M, pH and temperature not specified in the publication
0.654
penicillin G
-
mutant I305M/T213V, pH and temperature not specified in the publication
0.668
penicillin G
-
mutant I305M/T213V/S261M, pH and temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0038
penicillin G
-
recombinant wild-type enzyme, pH not specified in the publication, 30°C
0.004
penicillin G
-
wild-type enzyme, pH and temperature not specified in the publication
0.0055
penicillin G
-
recombinant mutant Q126M, pH not specified in the publication, 30°C
0.0058
penicillin G
-
recombinant mutant T213V, pH not specified in the publication, 30°C
0.009
penicillin G
-
recombinant mutant S261M, pH not specified in the publication, 30°C
0.0119
penicillin G
-
recombinant mutant S261A, pH not specified in the publication, 30°C
0.0171
penicillin G
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
0.144
penicillin G
-
mutant C155Y/Y184H/V275I/C281Y, pH and temperature not specified in the publication
0.176
penicillin G
-
mutant I305L, pH and temperature not specified in the publication
0.182
penicillin G
-
mutant I305M, pH and temperature not specified in the publication
0.203
penicillin G
-
mutant I305M/T213V, pH and temperature not specified in the publication
0.268
penicillin G
-
mutant I305M/T213V/S261M, pH and temperature not specified in the publication
0.307
penicillin G
-
mutantI305M/T213V/M73T, pH and temperature not specified in the publication
0.332
penicillin G
-
mutant I305M/S261M, pH and temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.203
-
recombinant enzyme, substrate ampicillin, determined by bioassay
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
160
-
recombinant mutant Y184A, pH not specified in the publication, 30°C
2.221
-
recombinant enzyme, substrate ampicillin, determined by HPLC
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
additional information
additional information
-
activity with different substrates and assay methods of recombinant wild-type and mutant enzymes, overview
additional information
activity with different substrates and assay methods of recombinant wild-type and mutant enzymes, overview
additional information
160% relative activity with 10 mM substrate penicillin G compared to the activity detected with 1 mM penicillin G
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
additional information
-
quaternary mutant (C155Y/Y184H/V275I/C281Y) with enhanced activity and without substrate inhibition
additional information
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
additional information
-
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
additional information
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
additional information
-
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
additional information
-
activity of recombinant wild-type and mutant enzymes with substrate penicillin G and penicillin N
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
36
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. The enzyme from Streptomyces clavuligerus shows an unusual ping-pong mechanism, which is significantly different from other members of the 2OG oxygenase superfamily
additional information
evolution
DAOCS is classified to the 2-oxoglutarate-Fe(II)-dioxygenase superfamily
evolution
the enzyme belongs to the iron- and 2-oxoglutarate-dependent oxidases
evolution
the enzyme is a member of the 2-oxoglutarate oxygenase superfamily, the members have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)-2OG-substrate ternary complex
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
determination of formation of a DAOCS·Fe(II)-2OG-penicillin N complex by nondenaturing ESI-MS analyses
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
34500
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE
34554
x * 34554, in solution the monomeric apoenzyme is in equilibrium with atrimeric form, electrospray mass spectrometry
30966
-
3 * 30966, DAOCS is in equilibrium with a trimeric form which is the form that crystallizes
34519
36000
60000
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
trimer
trimer
-
3 * 30966, DAOCS is in equilibrium with a trimeric form which is the form that crystallizes
additional information
?
x * 34554, in solution the monomeric apoenzyme is in equilibrium with atrimeric form, electrospray mass spectrometry
?
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE
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
additional information
active site structure in the apo-enzyme
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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 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
crystallization with 5-hydroxy-4-ketovaleric acid, 1.53 A resolution
crystallization with Fe(II) and succinate , 1.5 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), 2-oxoglutarate and ampicillin, 1.5 A resolution
crystallization with Fe(II), 2-oxoglutarate and penicillin G, 1.7 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
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
R258Q mutant, crystallization with Fe(II) and alpha-keto-beta-methylbutanoate, 1.5 and 1.6 A resolution
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
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
C155Y
C155Y/Y184H/S261M/V275I/C281Y/I305M
site-directed mutagenesis, the mutant shows 87fold increased kcat/Km for penicillin G compared to the wild-type
C155Y/Y184H/V275I/C281Y
C281F
substrate ampicillin, 191%, penicillin G, 264%, phenethicillin, 300%, and carbenicillin, 518% of wild-type activity
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
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
G79E
the mutant has 2.3fold increment in kcat/Km value when compared with the wild type enzyme
H244Q
I305L
I305M
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/C155Y/Y184H/T213V/V275I/C281Y/I305M
site-directed mutagenesis, the mutant shows 22fold increased specific activity and 81fold increased kcat/Km for penicillin G compared to the wild-type
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
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
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
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
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
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
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
R74L
mutation abolishes the activity of scDAOCS indicating the importance of these residue in binding the penicillin substrate
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/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
substrate ampicillin, 265%, penicillin G, 233%, phenethicillin, 330%, and carbenicillin,721% of wild-type activity
V275I/N304K
substrate ampicillin, 334%, penicillin G, 294%, phenethicillin, 635%, and carbenicillin, 867% of wild-type activity
V275I/N304L
substrate ampicillin, 225%, penicillin G, 141%, phenethicillin, 245%, and carbenicillin, 524% of wild-type activity
V275I/N304M
substrate ampicillin, 160%, penicillin G, 174%, phenethicillin, 261%, and carbenicillin, 230% of wild-type activity
V275I/N304R
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
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
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
A61E
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
C155Y/Y184H/V275I/C281Y
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
C281Y
C37S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
G79E
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to the wild-type enzyme
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
N304K
Q126M
R258A
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
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
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
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
R266L
-
2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present
R266Q
-
2-oxoglutarate conversion is very low and the same whether penicillin N, penicillin G or no penicillin substrate is present
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
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
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
V275I
V275I/C281Y/I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, over 32fold increased activity compared to the wild-type enzyme
V275I/I305M
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 32fold increased activity compared to the wild-type enzyme
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
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
additional information
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, quaternary mutant from error-prone PCR, the mutant shows 41fold increased kcat/Km for penicillin G compared to the wild-type
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 enzymes 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 enzymes catalytic effciency (101fold increment)
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
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
the mutant has 2.8fold increment in kcat/Km value when compared with the wild type enzyme
I305L
the mutant has 6.4fold increment in kcat/Km value when compared with the wild type enzyme
I305M
substrate ampicillin, 313%, penicillin G, 234%, phenethicillin, 318%, and carbenicillin, 450% of wild-type activity
I305M
the mutant has 10.8fold increment in kcat/Km value when compared with 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
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
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
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
substrate ampicillin, 270%, penicillin G, 237%, phenethicillin, 211%, an carbenicillin, 404% of wild-type activity
N304K
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
mutant enzyme shows increased penicillin analog 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
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
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
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
mutant enzyme with improved efficiency in penicillin conversion
R306L
substrate ampicillin, 186%, penicillin G, 105%, phenethicillin, 173%, and carbenicillin, 283% of wild-type activity
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
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
the mutant has 2.1fold increment in kcat/Km value when compared with 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
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
the mutant has 4.7fold increment in kcat/Km value when compared with the wild type enzyme
C281Y
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to 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
-
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
-
site-directed mutagenesis, the mutant shows increased activity with penicillin G compared to the wild-type enzyme
N304K
-
site-directed mutagenesis, exchange of residue surrounding the substrate, 6-14fold increased activity compared to the wild-type enzyme
Q126M
-
site-directed mutagenesis, the mutant shows activity increased to 281.5% with penicillin G compared to the wild-type
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
V275I
-
random mutagenesis, exchange of residue surrounding the substrate, 2-6fold increased activity compared to the wild-type enzyme
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
-
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
-
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
rational mutational approach to generate an engineered strain for enhanced production of from penicillin G, e.g. mutant FF8 from gene shuffling with 118fold increase in kcat/Km
additional information
reconstitution of TCA cycle with enzyme DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G, overview. Cumulative effects of different combinations ofDELTAsucA,DELTAaceA,DELTApoxB::acs, and DELTAampC mutations on G- 7-aminodeacetoxycephalosporanic acid (G-7-ADCA) production. Additional engineering steps are taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. Method optimization and evaluation, effects of deregulation of glyoxylate pathway and increased TCA flux on DAOCS catalyzed reaction, and effects of combining the beneficial mutations on G-7-ADCA production, overview
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5 - 9
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme expressed in Escherichia coli
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 enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli strain ESS by ion exchange chromatography and gel filtration
-
recombinant wild-type enzyme and mutant enzymes R258K, R258H, R258A, R258L and R258F
soluble recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 by anion exchange chromatography and gel filtration
-
wild-type and mutant enzymes R74I, R74Q, R75I/D270G, R75Q, R306L and R307Q
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloning and heterologous expression in Escherichia coli, Streptomyces lividans, Penicillium chrysogenum, Pseudomonas putida and Pichia pastoris, using of commonly used glutathione S-transferase to express DAOCS as a fusion protein
expressed in Escherichia coli, Streptomyces lividans, Penicillium chrysogenum, Pseudomonas putida, and Pichia pastoris
expression as glutathione S-transferase fusion protein in Echerichia coli
expression of wild-type and C-terminally truncated mutant enzymes as N-terminally His-tagged proteins in Escherichia coli strain BL21(DE3)
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
gene cefE, recombinant expression in Escherichia coli strain BW25311, functional recombinant expression of scDAOCS in Penicillium chrysogenum leading to expandase activity for penicillin N in the transfected cells. But only penicillin V is produced under the fermentation conditions, indicating that the industrial application of DAOCS is restricted by its low activities toward penicillin analogues other than penicillin N. In Penicillium chrysogenum strains expressing scDAOCS, a fermentation process is developed to produce adipyl-7-ADCA and adipyl-7-ACA, which can be converted to 7-ADCA and 7-ACA by glutaryl acylase. Recombinant expression of a hybrid enzyme,with parts from the enzymes of Streptomyces clavuligerus and Nocardia lactamdurans, in Streptomyces lividans strain W25, the recombinant enzyme is capable of converting penicillin G to G-7-aminodeacetoxycephalosporanic acid (G-7-ADCA). Recombinant expression of Streptomyces clavuligerus scDAOCS in a cefEF-disrupted cephalosporin C production Acremonium chrysogenum strain, which converts penicillin N accumulated in the disruption strain to deacetoxycephalosporin C
gene efeE, cloning in Escherichia coli strain DH5alpha, recombinant expression in Escherichia coli strain K12 (BW25113)
gene efeE, recombinant expression in Escherichia coli strain BL21(DE3)
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)
-
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 ESS
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
enzyme inactivated by 5,5-dithiobis-2-nitrobenzoic acid, complete reactivation by dithiothreitol
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
bioconversion of penicillins to cephalosporins using deacetoxycephalosporin C synthase (DAOCS) is an alternative and environmentally friendly process for production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), a key intermediate of many clinically useful semisynthetic cephalosporins
synthesis
-
enzyme produces the 7-aminodeacetoxycephalosporanic acid precursor utilized in industrial applications, optimization of enzyme activity for activity with the substrate penicillin G
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lee, H.J.; Lloyd, M.D.; Harlos, K.; Clifton, I.J.; Baldwin, J.E.; Schofield, C.J.
Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)
J. Mol. Biol.
308
937-948
2001
Streptomyces clavuligerus (P18548)
Valegard, K.; van Scheltinga, A.C.T.; Lloyd, M.D.; Hara, T.; Ramaswamy, S.; Perrakis, A.; Thompson, A.; Lee, H.J.; Baldwin, J.E.; Schofield, C.J.; Hajdu, J.; Andersson, I.
Structure of a cephalosporin synthase
Nature
394
805-809
1998
Streptomyces clavuligerus (P18548)
Yeh, W.K.; Ghag, S.K.; Queener, S.W.
Enzymes for epimerization of isopenicillin N, ring expansion of penicillin N, and 3'-hydroxylation of deacetoxycephalosporin C. Function, evolution, refolding, and enzyme engineering
Ann. N. Y. Acad. Sci.
672
396-408
1992
Acremonium chrysogenum, Streptomyces clavuligerus
-
Lee, H.J.; Dai, Y.F.; Shiau, C.Y.; Schofield, C.J.; Lloyd, M.D.
The kinetic properties of various R258 mutants of deacetoxycephalosporin C synthase
Eur. J. Biochem.
270
1301-1307
2003
Streptomyces clavuligerus, Streptomyces clavuligerus (P18548)
Lipscomb, S.J.; Lee, H.J.; Mukherji, M.; Baldwin, J.E.; Schofield, C.J.; Lloyd, M.D.
The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase
Eur. J. Biochem.
269
2735-2739
2002
Streptomyces clavuligerus
Lloyd, M.D.; Lee, H.J.; Harlos, K.; Zhang, Z.H.; Baldwin, J.E.; Schofield, C.J.; Charnock, J.M.; Garner, C.D.; Hara, T.; Terwisscha van Scheltinga, A.C.; Valeg.Ang.rd, K.; Viklund, J.A.C.; Hajdu, J.; Andersson, I.; Danielsson, A.; Bhikhabhai, R.
Studies on the active site of deacetoxycephalosporin C synthase
J. Mol. Biol.
287
943-960
1999
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Chin, H.S.; Sim, J.; Seah, K.I.; Sim, T.S.
Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity
FEMS Microbiol. Lett.
218
251-257
2003
Acremonium chrysogenum, Acremonium chrysogenum (Q9P4T5), Streptomyces clavuligerus, Streptomyces jumonjinensis, Streptomyces jumonjinensis (Q93FD4)
Kovacevic, S.; Weigel, B.J.; Tobin, M.B.; Ingolia, T.D.; Miller, J.R.
Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase
J. Bacteriol.
171
754-760
1989
Streptomyces clavuligerus
Sim, J.; Sim, T.S.
Mutational evidence supporting the involvement of tripartite residues His183, Asp185, and His243 in Streptomyces clavuligerus deacetoxycephalosporin C synthase for catalysis
Biosci. Biotechnol. Biochem.
64
828-832
2000
Streptomyces clavuligerus
Dotzlaf, J.E.; Yeh, W.K.
Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus
J. Biol. Chem.
264
10219-10227
1989
Streptomyces clavuligerus
Lubbe, C.; Wolfe, S.; Demain, A.L.
Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation
Enzyme Microb. Technol.
7
353-356
1985
Streptomyces clavuligerus
-
Chin, H.S.; Sim, T.S.
C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity
Biochem. Biophys. Res. Commun.
295
55-61
2002
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Chin, H.S.; Sim, J.; Sim, T.S.
Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analog conversion
Biochem. Biophys. Res. Commun.
287
507-513
2001
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Wei, C.L.; Yang, Y.B.; Wang, W.C.; Liu, W.C.; Hsu, J.S.; Tsai, Y.C.
Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G
Appl. Environ. Microbiol.
69
2306-2312
2003
Streptomyces clavuligerus
Oester, L.M.; van Scheltinga, A.C.; Valegard, K.; Hose, A.M.; Dubus, A.; Hajdu, J.; Andersson, I.
Conformational flexibility of the C terminus with implications for substrate binding and catalysis revealed in a new crystal form of deacetoxycephalosporin C synthase
J. Mol. Biol.
343
157-171
2004
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Jensen, S.E.; Westlake, D.W.S.; Wolfe, S.
Deacetoxycephalosporin C synthetase and deacetoxycephalosporin C hydroxylase are two separate enzymes in Streptomyces clavuligerus
J. Antibiot.
38
263-265
1985
Streptomyces clavuligerus
Baker, B.J.; Dotzlaf, J.E.; Yeh, W.K.
Deacetoxycephalosporin C hydroxylase of Streptomyces clavuligerus. Purification, characterization, bifunctionality, and evolutionary implication
J. Biol. Chem.
266
5087-5093
1991
Streptomyces clavuligerus
Wei, C.; Yang, Y.; Deng, C.; Liu, W.; Hsu, J.; Lin, Y.; Liaw, S.; Tsai, Y.
Directed evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion
Appl. Environ. Microbiol.
71
8873-8880
2005
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Goo, K.S.; Chua, C.S.; Sim, T.S.
Relevant double mutations in bioengineered Streptomyces clavuligerus deacetoxycephalosporin C synthase result in higher binding specificities which improve penicillin bioconversion
Appl. Environ. Microbiol.
74
1167-1175
2008
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Cicero, G.; Carbonera, C.; Valegard, K.; Hajdu, J.; Andersson, I.; Ranghino, G.
Study of the oxidative half-reaction catalyzed by a non-heme ferrous catalytic center by means of structural and computational methodologies
Int. J. Quantum Chem.
107
1514-1522
2007
Streptomyces clavuligerus (P18548)
-
Sim Goo, K.; Song Chua, C.; Sim, T.S.
A complete library of amino acid alterations at R306 in Streptomyces clavuligerus deacetoxycephalosporin C synthase demonstrates its structural role in the ring-expansion activity
Proteins
70
739-747
2008
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus
Goo, K.S.; Chua, C.S.; Sim, T.S.
Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production
J. Ind. Microbiol. Biotechnol.
36
619-633
2009
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus, Streptomyces clavuligerus NRRL 3585 (P18548)
Liras, P.; Demain, A.L.
Enzymology of beta-lactam compounds with cephem structure produced by actinomycete
Methods Enzymol.
458
401-429
2009
Actinomyces sp., Amycolatopsis lactamdurans, Streptomyces clavuligerus, Amycolatopsis lactamdurans NRRL3802, Streptomyces clavuligerus ATCC 27064
Chin, H.; Goh, K.; Teo, K.; Chan, M.; Lee, S.; Ong, L.
Predicting the catalytic sites of Streptomyces clavuligerus deacetylcephalosporin C synthase and clavaminate synthase 2
Afr. J. Microbiol. Res.
5
3357-3366
2011
Streptomyces clavuligerus (P18548)
-
Ji, J.; Fan, K.; Tian, X.; Zhang, X.; Zhang, Y.; Yang, K.
Iterative combinatorial mutagenesis as an effective strategy for generation of deacetoxycephalosporin C synthase with improved activity toward penicillin G
Appl. Environ. Microbiol.
78
7809-7812
2012
Streptomyces clavuligerus
Ji, J.; Tian, X.; Fan, K.; Yang, K.
New strategy of site-directed mutagenesis identifies new sites to improve Streptomyces clavuligerus deacetoxycephalosporin C synthase activity toward penicillin G
Appl. Microbiol. Biotechnol.
93
2395-2401
2012
Streptomyces clavuligerus, Streptomyces clavuligerus ATCC 27064
Tarhonskaya, H.; Szoelloessi, A.; Leung, I.K.; Bush, J.T.; Henry, L.; Chowdhury, R.; Iqbal, A.; Claridge, T.D.; Schofield, C.J.; Flashman, E.
Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases
Biochemistry
53
2483-2493
2014
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus ATCC 27064 (P18548)
Fan, K.; Lin, B.; Tao, Y.; Yang, K.
Engineering deacetoxycephalosporin C synthase as a catalyst for the bioconversion of penicillins
J. Ind. Microbiol. Biotechnol.
44
705-710
2017
Acremonium chrysogenum (P11935), Streptomyces clavuligerus (P18548), Amycolatopsis lactamdurans (Q03047), Streptomyces clavuligerus ATCC 27064 (P18548)
Lin, B.; Fan, K.; Zhao, J.; Ji, J.; Wu, L.; Yang, K.; Tao, Y.
Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G
Proc. Natl. Acad. Sci. USA
112
9855-9859
2015
Streptomyces clavuligerus (P18548), Streptomyces clavuligerus ATCC 27064 (P18548)
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