BRENDA - Enzyme Database show
show all sequences of 1.14.11.21

Expression and purification of two isoenzymes of clavaminate synthase and initial characterization of the iron binding site. General error analysis in polymerase chain reaction amplification

Busby, R.W.; Chang, M.D.T.; Busby, R.C.; Wimp, J.; Townsend, C.A.; J. Biol. Chem. 270, 4262-4269 (1995)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
isozymes CS1 and CS2, overexpression in Escherichia coli JM101
Streptomyces clavuligerus
General Stability
General Stability
Organism
Co2+ completely inhibits the enzyme, competitive to Fe2+, but also stabilizes the enzyme against self-inactivation in absence of proclavaminate
Streptomyces clavuligerus
Inhibitors
Inhibitors
Commentary
Organism
Structure
Co2+
complete inhibition competitive to Fe2+, but stabilizes the enzyme against self-inactivation in absence of proclavaminate
Streptomyces clavuligerus
diethyl dicarbonate
isozyme CS2, rapid inactivation at pH 6.0, 25°C, t1/2: 1.1 min
Streptomyces clavuligerus
N-ethylmaleimide
inactivation
Streptomyces clavuligerus
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.13
-
proclavaminate
isozyme CS2, pH 7.0, 22°C
Streptomyces clavuligerus
0.22
-
proclavaminate
isozyme CS1, pH 7.0, 22°C
Streptomyces clavuligerus
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Fe2+
required
Streptomyces clavuligerus
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
35360
-
CS1, mass spectrometry; recombinant enzyme
Streptomyces clavuligerus
35750
-
CS2, mass spectrometry; recombinant enzyme
Streptomyces clavuligerus
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
Streptomyces clavuligerus
-
guanidinoproclavaminate + succinate + CO2 + H2O
-
Streptomyces clavuligerus
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Streptomyces clavuligerus
-
2 isozymes CS1 and CS2
-
Oxidation Stability
Oxidation Stability
Organism
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
Streptomyces clavuligerus
Purification (Commentary)
Commentary
Organism
recombinant isozymes CS1 and CS2 to homogeneity, 10fold
Streptomyces clavuligerus
Reaction
Reaction
Commentary
Organism
dihydroclavaminate + 2-oxoglutarate + O2 = clavaminate + succinate + CO2 + H2O
reaction mechanism
Streptomyces clavuligerus
proclavaminate + 2-oxoglutarate + O2 = dihydroclavaminate + succinate + CO2 + H2O
reaction mechanism
Streptomyces clavuligerus
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.8
-
purified recombinant isozyme CS2
Streptomyces clavuligerus
0.86
-
purified recombinant isozyme CS1
Streptomyces clavuligerus
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
-
639275
Streptomyces clavuligerus
guanidinoproclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
hydroxylation
639275
Streptomyces clavuligerus
guanidinoproclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
dihydroclavaminate + 2-oxoglutarate + O2
cyclization
639275
Streptomyces clavuligerus
clavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
additional information
1 or more histidine as well as cysteine residue located at or near the active site or in the metal binding site, critical for catalysis
639275
Streptomyces clavuligerus
?
-
-
-
-
additional information
dependent on O2 and 2-oxoglutarate
639275
Streptomyces clavuligerus
?
-
-
-
-
proclavaminate + 2-oxoglutarate + O2
saturation
639275
Streptomyces clavuligerus
dihydroclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Streptomyces clavuligerus
Cloned(Commentary) (protein specific)
Commentary
Organism
isozymes CS1 and CS2, overexpression in Escherichia coli JM101
Streptomyces clavuligerus
General Stability (protein specific)
General Stability
Organism
Co2+ completely inhibits the enzyme, competitive to Fe2+, but also stabilizes the enzyme against self-inactivation in absence of proclavaminate
Streptomyces clavuligerus
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
Co2+
complete inhibition competitive to Fe2+, but stabilizes the enzyme against self-inactivation in absence of proclavaminate
Streptomyces clavuligerus
diethyl dicarbonate
isozyme CS2, rapid inactivation at pH 6.0, 25°C, t1/2: 1.1 min
Streptomyces clavuligerus
N-ethylmaleimide
inactivation
Streptomyces clavuligerus
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.13
-
proclavaminate
isozyme CS2, pH 7.0, 22°C
Streptomyces clavuligerus
0.22
-
proclavaminate
isozyme CS1, pH 7.0, 22°C
Streptomyces clavuligerus
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Fe2+
required
Streptomyces clavuligerus
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
35360
-
CS1, mass spectrometry; recombinant enzyme
Streptomyces clavuligerus
35750
-
CS2, mass spectrometry; recombinant enzyme
Streptomyces clavuligerus
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
Streptomyces clavuligerus
-
guanidinoproclavaminate + succinate + CO2 + H2O
-
Streptomyces clavuligerus
?
Oxidation Stability (protein specific)
Oxidation Stability
Organism
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
Streptomyces clavuligerus
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant isozymes CS1 and CS2 to homogeneity, 10fold
Streptomyces clavuligerus
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.8
-
purified recombinant isozyme CS2
Streptomyces clavuligerus
0.86
-
purified recombinant isozyme CS1
Streptomyces clavuligerus
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
-
639275
Streptomyces clavuligerus
guanidinoproclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
deoxyguanidinoproclavaminate + 2-oxoglutarate + O2
hydroxylation
639275
Streptomyces clavuligerus
guanidinoproclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
dihydroclavaminate + 2-oxoglutarate + O2
cyclization
639275
Streptomyces clavuligerus
clavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
additional information
1 or more histidine as well as cysteine residue located at or near the active site or in the metal binding site, critical for catalysis
639275
Streptomyces clavuligerus
?
-
-
-
-
additional information
dependent on O2 and 2-oxoglutarate
639275
Streptomyces clavuligerus
?
-
-
-
-
proclavaminate + 2-oxoglutarate + O2
saturation
639275
Streptomyces clavuligerus
dihydroclavaminate + succinate + CO2 + H2O
-
639275
Streptomyces clavuligerus
?
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Streptomyces clavuligerus
Other publictions for EC 1.14.11.21
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
742159
Shrestha
-
Heterologous production of cl ...
Streptomyces clavuligerus
Biotechnol. Bioprocess Eng.
22
359-365
2017
-
1
1
-
-
-
-
-
-
-
-
2
-
1
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2
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2
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1
1
-
-
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723902
Chin
-
Predicting the catalytic sites ...
Streptomyces clavuligerus
Afr. J. Microbiol. Res.
5
3357-3366
2011
-
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1
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2
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-
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-
2
4
-
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-
685142
Borowski
Mechanism for cyclization reac ...
Streptomyces clavuligerus
Biochemistry
46
3682-3691
2007
-
-
-
1
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1
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1
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639036
Townsend
New reactions in clavulanic ac ...
Streptomyces clavuligerus
Curr. Opin. Chem. Biol.
6
583-589
2002
-
-
-
-
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1
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4
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1
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1
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9
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1
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4
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9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
639286
Zhang
Crystal structure of a clavami ...
Streptomyces clavuligerus
FEBS Lett.
517
7-12
2002
-
-
-
1
-
-
-
-
-
1
-
4
-
1
-
-
-
3
-
-
-
-
9
-
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1
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1
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4
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-
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-
9
-
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-
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-
-
-
-
-
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639284
Zhou
Spectroscopic studies of subst ...
Streptomyces clavuligerus
J. Am. Chem. Soc.
123
7388-7398
2001
-
-
-
-
-
-
-
-
-
1
-
4
-
1
-
-
-
2
-
-
-
-
11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
11
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
639281
Zhang
Structural origins of the sele ...
Streptomyces clavuligerus
Nat. Struct. Biol.
7
127-133
2000
-
-
-
1
-
-
-
-
-
1
-
-
-
1
-
-
-
2
-
-
-
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8
-
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-
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-
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-
1
-
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1
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8
-
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-
-
-
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-
-
-
639282
Khaleeli
Site-directed mutagenesis and ...
Streptomyces clavuligerus
Biochemistry
39
8666-8673
2000
-
-
1
-
8
-
-
-
-
1
-
-
-
1
-
-
1
-
-
-
-
-
3
-
1
-
-
-
1
-
-
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-
-
-
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-
1
-
-
8
-
-
-
-
-
-
1
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-
1
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3
-
1
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-
1
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-
-
-
639283
Doan
Mutagenesis studies on the iro ...
Streptomyces clavuligerus
Biochem. Biophys. Res. Commun.
279
240-244
2000
-
-
-
-
15
-
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1
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1
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2
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5
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15
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1
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5
-
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-
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-
-
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-
639280
Lloyd
-
Product-substrate engineering ...
Streptomyces clavuligerus
Tetrahedron
55
10201-10220
1999
-
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1
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4
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1
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3
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11
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1
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4
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11
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639279
Zhou
-
Substrate binding to the alpha ...
Streptomyces clavuligerus
J. Am. Chem. Soc.
120
13539-13540
1998
-
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1
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4
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1
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9
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1
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9
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639278
Baldwin
-
Chemo-enzymic synthesis of bic ...
Streptomyces clavuligerus
Tetrahedron
53
7011-7020
1997
-
1
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1
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4
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1
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9
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1
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639277
Busby
A single monomeric iron center ...
Streptomyces clavuligerus
Bioorg. Med. Chem.
4
1059-1064
1996
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1
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2
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1
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5
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1
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3
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11
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1
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1
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2
1
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1
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5
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11
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639274
Janc
Purification and characterizat ...
Streptomyces antibioticus, Streptomyces antibioticus Tu1718, Streptomyces clavuligerus
J. Biol. Chem.
270
5399-5404
1995
-
-
-
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-
-
3
-
2
6
15
-
5
1
-
1
6
-
-
1
1
31
2
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
2
6
15
-
1
-
1
-
-
1
1
31
2
-
-
-
-
2
-
-
-
-
-
-
-
-
-
639275
Busby
Expression and purification of ...
Streptomyces clavuligerus
J. Biol. Chem.
270
4262-4269
1995
-
-
1
-
-
1
3
2
-
1
2
1
-
3
1
-
1
2
-
-
2
-
6
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
1
-
3
-
2
-
1
2
1
-
1
-
1
-
-
2
-
6
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
639276
Paradkar
Functional analysis of the gen ...
Streptomyces clavuligerus, Streptomyces clavuligerus NRRL 3585
J. Bacteriol.
177
1307-1314
1995
-
-
1
-
1
-
-
-
-
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-
9
-
5
-
-
-
3
-
-
1
-
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
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-
-
9
-
-
-
-
-
-
1
-
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
639273
Iwata-Reuyl
Synthesis and reaction of pote ...
Streptomyces clavuligerus
J. Nat. Prod.
56
1373-1396
1993
-
-
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11
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1
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11
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1
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