Information on EC 1.14.11.21 - clavaminate synthase

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The expected taxonomic range for this enzyme is: Streptomyces

EC NUMBER
COMMENTARY hide
1.14.11.21
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RECOMMENDED NAME
GeneOntology No.
clavaminate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
deoxyamidinoproclavaminate + 2-oxoglutarate + O2 = amidinoproclavaminate + succinate + CO2
show the reaction diagram
dihydroclavaminate + 2-oxoglutarate + O2 = clavaminate + succinate + CO2 + H2O
show the reaction diagram
proclavaminate + 2-oxoglutarate + O2 = dihydroclavaminate + succinate + CO2 + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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oxidative cyclization
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-
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oxidative decarboxylation
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-
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redox reaction
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-
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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clavulanate biosynthesis
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Clavulanic acid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
deoxyamidinoproclavaminate,2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating)
Contains nonheme iron. Catalyses three separate oxidative reactions in the pathway for the biosythesis of the beta-lactamase inhibitor clavulanate in Streptomyces clavuligerus. The first step (hydroxylation) is separated from the latter two (oxidative cyclization and desaturation) by the action of EC 3.5.3.22, proclavaminate amidinohydrolase. The three reactions are all catalysed at the same nonheme iron site.
CAS REGISTRY NUMBER
COMMENTARY hide
122799-56-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain Tü 1718, CS3, no production of clavulanic acid
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Manually annotated by BRENDA team
strain Tü 1718, CS3, no production of clavulanic acid
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Manually annotated by BRENDA team
strain NRRL 3585
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
CAS1 is classified to the taurine catabolism dioxygenase TauD family with the Pfam accession of PF02668, referred to as subgroup II of the 2-oxoglutarate-Fe(II)-dioxygenase superfamily; CAS2 is classified to the taurine catabolism dioxygenase TauD family with the Pfam accession of PF02668, referred to as subgroup II of the 2-oxoglutarate-Fe(II)-dioxygenase superfamily
additional information
structure simulations, docking, and modeling using the amino acid sequence CAS2, active site prediction, overview; structure simulations. docking, and modeling using the amino acid sequence of CAS1, active site prediction, residue R258 is involved in 2-oxoglutarate binding, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxoglutarate + O2
? + H2O2
show the reaction diagram
deoxyamidinoproclavaminate + 2-oxoglutarate + O2
amidinoproclavaminate + succinate + CO2 + H2O
show the reaction diagram
deoxyamidinoproclavaminate + 2-oxoglutarate + O2
E-(2S)-5-amino-2(2'-oxoazetidin-1-yl)-pent-3,4-enoate + succinate + CO2 + H2O
show the reaction diagram
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isozyme CS2
10% of the product is proclavaminate, 90% of the product is E-(2S)-5-amino-2(2'-oxoazetidin-1-yl)-pent-3,4-enoate
?
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
guanidinoproclavaminate + succinate + CO2 + H2O
show the reaction diagram
dihydroclavaminate + 2-oxoglutarate + O2
clavaminate + succinate + CO2 + H2O
show the reaction diagram
N-alpha-acetyl-L-arginine + 2-oxoglutarate + O2
4-hydroxy-N-alpha-acetyl-L-arginine + succinate + CO2 + H2O
show the reaction diagram
proclavaminate + 2-oxoglutarate + O2
dihydroclavaminate + succinate + CO2 + H2O
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
deoxyamidinoproclavaminate + 2-oxoglutarate + O2
amidinoproclavaminate + succinate + CO2 + H2O
show the reaction diagram
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
guanidinoproclavaminate + succinate + CO2 + H2O
show the reaction diagram
dihydroclavaminate + 2-oxoglutarate + O2
clavaminate + succinate + CO2 + H2O
show the reaction diagram
proclavaminate + 2-oxoglutarate + O2
dihydroclavaminate + succinate + CO2 + H2O
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4'-ethynyl proclavaminate
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noncompetitive
4'-vinyl proclavaminate
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noncompetitive
4-cyclopropyl proclavaminate
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noncompetitive
ascorbate
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inactivation due to release of peroxide from reaction of ascorbate and O2 in absence of proclavaminate, but in presence of Fe2+, t1/2: 50 min, catalase protects
diethyl dicarbonate
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isozyme CS2, rapid inactivation at pH 6.0, 25°C, t1/2: 1.1 min
N-bromoacetyl-L-arginine
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isozyme CS2, inactivation, at 7.5 mM half-life of 11.2 min at 30°C, Co2+ competes, substrates protect
N-ethylmaleimide
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inactivation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19 - 0.38
(2S,3R)-proclavaminate
0.039 - 0.042
2-oxoglutarate
0.0019 - 0.0032
Fe2+
0.23
N-alpha-acetyl-L-arginine
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pH 7.0, 22°C
0.13 - 0.22
proclavaminate
0.38
rac-proclavaminate
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pH 7.0, 22°C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.765
(2S,3R)-proclavaminate
Streptomyces clavuligerus
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pH 7.0, 40°C
6.4
N-alpha-acetyl-L-arginine
Streptomyces clavuligerus
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pH 7.0, 22°C
0.725
rac-proclavaminate
Streptomyces clavuligerus
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pH 7.0, 40°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
38.3
N-bromoacetyl-L-arginine
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isozyme CS2, pH 7.0, 30°C
additional information
additional information
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Ki of 4'-vinyl proclavaminate, 4'-ethynyl proclavaminate, and 4-cyclopropyl proclavaminate is in the range of 2-10 mM
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.1
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purified enzyme
0.4
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65fold purified enzyme
0.57
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purified enzyme, 40°C
0.8
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purified recombinant isozyme CS2
0.86
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purified recombinant isozyme CS1
additional information
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activity of wild-type and mutant in different growth media
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
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room temperature, assay at
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)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35840
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CS3, mass spectrometry
38000
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CS1, gel filtration
41000
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CS3, gel filtration
42000
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CS2, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anaerobically, recombinant isozyme CAS1, in complex with Fe2+, 2-oxoglutarate and deoxyguanidinoproclavaminate, hanging drop vapour diffusion method, protein solution: 20 mg/ml, reservoir solution: 0.1 M Tris, pH 7.5, 2 M ammonium sulfate, 10% glycerol, 20°C, treatment with NO under anaerobic conditions, structure determination and analysis
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moelcular dynamics simulation, model of the active site
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recombinant isozyme CAS1, hanging drop method, 2 M ammonium sulfate, 0.1 M Tris, 21°C, apoform crystals or in complex with Fe2+, N-alpha-acetyl-L-arginine, and the substrates 2-oxoglutarate and proclavaminate, X-ray diffraction structure determination for native and heavy atom derivatives and analysis
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Co2+ completely inhibits the enzyme, competitive to Fe2+, but also stabilizes the enzyme against self-inactivation in absence of proclavaminate
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
inactivation due to release of peroxide from reaction of 2-oxoglutarate and O2 in absence of proclavaminate, in presence of Fe2+, t1/2: 5 min, with ascorbate instead of 2-oxoglutarate t1/2: 50 min, catalase protects
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639270, 639275
inactivation due to release of peroxide from reaction of 2-oxoglutarate and O2 in absence of proclavaminate, in presence of Fe2+, t1/2: 52 s, catalase partially protects
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639274
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, purified enzyme, frozen in liquid N2, stable for at least several months
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-80°C, purified protein, 50 mM MOPS, pH 7.0, 0.02 mM EDTA, 2 mM DTT, 50% glycerol, stable for at least 1 year
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
12.5fold to homogeneity
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recombinant isozymes CS1 and CS2 to homogeneity, 10fold
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recombinant wild-type and mutants of isozyme CS2 from Escherichia coli JM101
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene cs2, isozyme CS2, wild-type and mutants, overexpression in Escherichia coli JM101
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isozymes CAS1 and CAS2 encoded by genes cas1 and cas2, respectively, genetic analysis
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isozymes CS1 and CS2, overexpression in Escherichia coli JM101
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C162L
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isozyme CAS2, site-directed mutagenesis, same activity in hydroxylation of N-alpha-acetyl-L-arginine as the wild-type enzyme
C200L
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isozyme CAS2, site-directed mutagenesis, 42% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
C201L
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isozyme CAS2, site-directed mutagenesis, 40% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
C8L
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isozyme CAS2, site-directed mutagenesis, 42% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H109L
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isozyme CAS2, site-directed mutagenesis, 86% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H109Q
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isozyme CS2, site-directed mutagensis, 83% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
H122L
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isozyme CAS2, site-directed mutagenesis, below 1% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H122Q
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isozyme CS2, site-directed mutagensis, 96% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
H131L
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isozyme CAS2, site-directed mutagenesis, 36% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H131Q
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isozyme CS2, site-directed mutagensis, 16% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
H145E
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isozyme CAS2, site-directed mutagenesis, below 1% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H145L
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isozyme CAS2, site-directed mutagenesis, below 1% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H151l
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isozyme CAS2, site-directed mutagenesis, below 1% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H151Q
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isozyme CS2, site-directed mutagensis, 10% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
H167L
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isozyme CAS2, site-directed mutagenesis, 19% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H167Q
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isozyme CS2, site-directed mutagensis, 95% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
H280L
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isozyme CAS2, site-directed mutagenesis, 86% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H280Q
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isozyme CS2, site-directed mutagensis, no activity of clavaminate formation, but slight hydroxylating activity of deoxyguanidinoproclavaminate
H297L
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isozyme CAS2, site-directed mutagenesis, 3% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
H297Q
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isozyme CS2, site-directed mutagensis, 65% activity of clavaminate formation compared to the wild-type enzyme, unaltered hydroxylating activity
Q154L
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isozyme CAS2, site-directed mutagenesis, 23% activity in hydroxylation of N-alpha-acetyl-L-arginine compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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chemo-enzymatic synthesis of bicyclic gamma-lactams using recombinant purified CAS2 isozyme, reaction mechanism