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show all sequences of 1.13.11.50

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

Leitgeb, S.; Straganz, G.D.; Nidetzky, B.; Biochem. J. 418, 403-411 (2009)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expression of mutant gene vector in Escherichia coli BL21(DE3)
Acinetobacter johnsonii
Engineering
Amino acid exchange
Commentary
Organism
H104E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H104N
approximately 1% of the specific activity of wild-type enzyme with substrate pentan-2,4-dione, retains binding affinity for Fe2+ at binding site I, binding seems to be tighter than in wild-type, small effect on other metal ions
Acinetobacter johnsonii
H62E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H62N
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, binding disruption of Cu2+, Mn2+, and Ni2+ compared with wild-type
Acinetobacter johnsonii
H64D
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H64E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H64N
conversion of substrate in a strictly Fe2+-concentration dependent manner, substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
additional information
the exchange of 3 histidines in the Fe2+-binding centre shows that these histidines are crucial for for binding Fe2+ and for Fe2+-dependent dioxygenase activity
Acinetobacter johnsonii
Inhibitors
Inhibitors
Commentary
Organism
Structure
Cu2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
H2O2
1 M H2O2 causes complete loss of enzyme activity in less than 10 min, contains no Fe2+ (probably oxidized to Fe3+), partial reconstitution (40%) with 2 mM Fe2+, 20 mM Tris/HCl buffer, pH 7.5, 25°C
Acinetobacter johnsonii
Mn2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
additional information
no inactivation effect of Fe3+
Acinetobacter johnsonii
Ni2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
Zn2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enzyme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Cu2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Fe2+
sulfate, necessary for enzyme activity. About 0.9 mol Fe2+/mol wild-type enzyme, less than 5% Fe2+ in mutants except 0.27 mol/mol H104E-enzyme and 0.45 mol/mol H104N-enzyme
Acinetobacter johnsonii
Fe3+
citrate, no interference with Fe2+
Acinetobacter johnsonii
Mn2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Ni2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Zn2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
18000
-
SDS-PAGE
Acinetobacter johnsonii
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
pentane-2,4-dione + O2
Acinetobacter johnsonii
-
methylglyoxal + acetate
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Acinetobacter johnsonii
Q8GNT2
-
-
Purification (Commentary)
Commentary
Organism
purified, concentrated enzyme is dialyzed twice against 2 mM EDTA in 20 mM Tris/HCl (pH 7.5) to strip off iron, incubation in 2 mM metal ion as sulfate salt plus ascorbate to prevent Fe2+ oxidation, unbound metal ions are removed by 3 cycles of gel filtration using nucleic acid purification column
Acinetobacter johnsonii
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.09
-
wild-type enzyme, 20 mM Tris/HCl buffer, pH 7.5, 25°C
Acinetobacter johnsonii
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3,4-dihydroxyphenylacetate + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
pentane-2,4-dione + O2
-
696107
Acinetobacter johnsonii
methylglyoxal + acetate
-
-
-
?
potassium oxalate + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
quercetin + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
Subunits
Subunits
Commentary
Organism
homotetramer
-
Acinetobacter johnsonii
Cofactor
Cofactor
Commentary
Organism
Structure
heme
necessary for enzyme activity
Acinetobacter johnsonii
Cloned(Commentary) (protein specific)
Commentary
Organism
expression of mutant gene vector in Escherichia coli BL21(DE3)
Acinetobacter johnsonii
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
heme
necessary for enzyme activity
Acinetobacter johnsonii
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
H104E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H104N
approximately 1% of the specific activity of wild-type enzyme with substrate pentan-2,4-dione, retains binding affinity for Fe2+ at binding site I, binding seems to be tighter than in wild-type, small effect on other metal ions
Acinetobacter johnsonii
H62E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H62N
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, binding disruption of Cu2+, Mn2+, and Ni2+ compared with wild-type
Acinetobacter johnsonii
H64D
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H64E
no activity in initial rate assays with substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
H64N
conversion of substrate in a strictly Fe2+-concentration dependent manner, substrate pentan-2,4-dione, no binding of Fe2+ at binding site I, small effect on other metal ions
Acinetobacter johnsonii
additional information
the exchange of 3 histidines in the Fe2+-binding centre shows that these histidines are crucial for for binding Fe2+ and for Fe2+-dependent dioxygenase activity
Acinetobacter johnsonii
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
Cu2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
H2O2
1 M H2O2 causes complete loss of enzyme activity in less than 10 min, contains no Fe2+ (probably oxidized to Fe3+), partial reconstitution (40%) with 2 mM Fe2+, 20 mM Tris/HCl buffer, pH 7.5, 25°C
Acinetobacter johnsonii
Mn2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
additional information
no inactivation effect of Fe3+
Acinetobacter johnsonii
Ni2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enyzme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
Zn2+
20 mM Tris/HCl buffer, pH 7.5, 25°C, 1.2fold molar excess, reversible inactivation of wild-type and mutant enzyme through competition with Fe2+, substrates 200 microM pentane-2,4-dione, 330 microM quercetin, 330 microM potassium oxalate, 330 microM 3,4-dihydroxyphenylacetate
Acinetobacter johnsonii
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Cu2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Fe2+
sulfate, necessary for enzyme activity. About 0.9 mol Fe2+/mol wild-type enzyme, less than 5% Fe2+ in mutants except 0.27 mol/mol H104E-enzyme and 0.45 mol/mol H104N-enzyme
Acinetobacter johnsonii
Fe3+
citrate, no interference with Fe2+
Acinetobacter johnsonii
Mn2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Ni2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Zn2+
sulfate, similar binding as Fe2+ in wild-type enzyme
Acinetobacter johnsonii
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
18000
-
SDS-PAGE
Acinetobacter johnsonii
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
pentane-2,4-dione + O2
Acinetobacter johnsonii
-
methylglyoxal + acetate
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
purified, concentrated enzyme is dialyzed twice against 2 mM EDTA in 20 mM Tris/HCl (pH 7.5) to strip off iron, incubation in 2 mM metal ion as sulfate salt plus ascorbate to prevent Fe2+ oxidation, unbound metal ions are removed by 3 cycles of gel filtration using nucleic acid purification column
Acinetobacter johnsonii
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
0.09
-
wild-type enzyme, 20 mM Tris/HCl buffer, pH 7.5, 25°C
Acinetobacter johnsonii
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3,4-dihydroxyphenylacetate + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
pentane-2,4-dione + O2
-
696107
Acinetobacter johnsonii
methylglyoxal + acetate
-
-
-
?
potassium oxalate + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
quercetin + O2
-
696107
Acinetobacter johnsonii
?
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homotetramer
-
Acinetobacter johnsonii
Other publictions for EC 1.13.11.50
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)
742322
Brkic
-
Insight of the iron binding a ...
Acinetobacter johnsonii
Croat. Chem. Acta
88
297-306
2015
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-
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1
3
-
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1
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1
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1
3
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1
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725553
Brkic
Dke1--structure, dynamics, and ...
Acinetobacter johnsonii
J. Biol. Inorg. Chem.
17
801-815
2012
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-
1
-
4
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1
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2
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1
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1
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1
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2
1
1
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10
1
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1
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4
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-
1
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2
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1
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-
1
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2
1
1
-
-
10
1
-
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-
2
2
-
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-
725198
Diebold
Spectroscopic and computationa ...
Acinetobacter johnsonii
J. Am. Chem. Soc.
133
15979-15991
2011
-
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1
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2
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1
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3
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1
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3
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1
1
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711258
Diebold
The three-his triad in Dke1: c ...
Acinetobacter johnsonii
Biochemistry
49
6945-6952
2010
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-
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2
-
1
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1
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2
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2
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1
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2
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-
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-
711267
Straganz
Kinetic and CD/MCD spectroscop ...
Acinetobacter johnsonii
Biochemistry
49
996-1004
2010
-
-
-
-
9
-
-
1
-
1
-
1
-
1
-
-
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-
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3
-
1
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-
10
1
1
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-
9
-
-
-
-
1
-
1
-
1
-
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-
-
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-
3
-
1
-
-
10
1
1
-
-
-
-
-
-
-
-
696107
Leitgeb
Biochemical characterization a ...
Acinetobacter johnsonii
Biochem. J.
418
403-411
2009
-
-
1
-
8
-
6
-
-
6
1
1
-
2
-
-
1
-
-
-
1
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4
1
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1
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1
1
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8
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6
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6
1
1
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1
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1
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4
1
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675466
Straganz
-
Exploring the cupin-type metal ...
Acinetobacter johnsonii
J. Mol. Catal. B
39
171-178
2006
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-
1
-
1
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2
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1
1
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1
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1
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3
1
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1
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1
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3
1
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657614
Hofer
Fast determination of operatio ...
Acinetobacter johnsonii
Appl. Microbiol. Biotechnol.
1
1-12
2005
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1
1
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1
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2
1
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1
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1
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1
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1
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1
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1
1
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1
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657866
Grogan
Emergent mechanistic diversity ...
Acinetobacter johnsonii
Biochem. J.
388
721-730
2005
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-
1
1
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1
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1
1
1
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1
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1
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6
1
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1
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1
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1
1
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1
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6
1
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1
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671683
Straganz
-
Integrated approach for produc ...
Acinetobacter johnsonii
Biocatal. Biotransform.
23
261-269
2005
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-
1
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3
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1
4
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1
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1
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1
1
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3
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1
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1
4
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1
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1
1
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3
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674150
Straganz
Reaction coordinate analysis f ...
Acinetobacter johnsonii
J. Am. Chem. Soc.
127
12306-12314
2005
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4
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1
2
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3
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1
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4
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4
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1
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4
1
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4
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636327
Straganz
Acetylacetone-cleaving enzyme ...
Acinetobacter johnsonii
Biochem. J.
369
573-581
2003
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-
1
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5
1
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1
1
1
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3
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1
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11
1
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1
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1
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5
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1
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1
1
1
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1
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11
1
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1
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636328
Straganz
A novel beta-diketone-cleaving ...
Acinetobacter johnsonii
Biochem. Biophys. Res. Commun.
297
232-236
2002
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2
1
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1
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5
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-
-
2
1
-
-
-
-
1
-
-
2
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-