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show all sequences of 1.3.99.32

Structural basis for promoting and preventing decarboxylation in glutaryl-coenzyme A dehydrogenases

Wischgoll, S.; Demmer, U.; Warkentin, E.; Guenther, R.; Boll, M.; Ermler, U.; Biochemistry 49, 5350-5357 (2010)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expression of His-tagged GDHDes in Escherichia coli
Desulfococcus multivorans
Crystallization (Commentary)
Crystallization
Organism
purified recombinant His-tagged GDHDes, hanging drop vapor diffusion method, 20 mg/ml protein in 10 mM MES, pH 6.0, 0.5 M KCl, 10% w/v glycerol, 1 mM DTT, 1 mM FAD, and 2 mM glutaryl-CoA are mixed with an equal volume of reservoir solution containing 50% v/v MPD, 0.1 M Tris-HCl, pH 8.5, and 0.2 M NH4H2PO4, at 4°C, X-ray diffraction structure determination and analysis
Desulfococcus multivorans
Engineering
Amino acid exchange
Commentary
Organism
A80E
site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
A80E/S161T/V366Y
site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
A80E/V366Y
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
V366Y
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
V88S
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.014
-
glutaryl-CoA
pH 7.8, 30°C, mutant A80E/V366Y
Desulfococcus multivorans
0.028
-
glutaryl-CoA
pH 7.8, 30°C, mutant V366Y
Desulfococcus multivorans
0.053
-
glutaryl-CoA
pH 7.8, 30°C, wild-type enzyme
Desulfococcus multivorans
0.059
-
glutaryl-CoA
pH 7.8, 30°C, mutant V88S
Desulfococcus multivorans
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
glutaryl-CoA + FAD
Desulfococcus multivorans
-
glutaconyl-CoA + FADH2
-
-
?
additional information
Desulfococcus multivorans
the decarboxylating, EC 1.3.99.7, and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr to Val exchange strictly conserved between the two GDH types, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved Arg is stronger in GDHDes than in the decarboxylating enzyme, molecular dynamics. The identified structural changes prevent decarboxylation 1. by strengthening the C4-C5 bond of glutaconyl-CoA, 2. by reducing the leaving group potential of CO2, and 3. by increasing the distance between the C4 atom, negatively charged in the dienolate transition state, and the adjacent glutamic acid
?
-
-
-
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Desulfococcus multivorans
C3UVB0
-
-
Purification (Commentary)
Commentary
Organism
recombinant His-tagged GDHDes from Escherichia coli by nickel affinity chromatography
Desulfococcus multivorans
Reaction
Reaction
Commentary
Organism
glutaryl-CoA + acceptor = (E)-glutaconyl-CoA + reduced acceptor
the structure of the GDHDes-glutaconyl-CoA complex is completely compatible with the mechanism of the reductive half-reaction established for acyl-CoA dehydrogenases, which is characterized by the rupture of two kinetically stable C-H bonds. The substrate binds to the re side of the FAD ring, and the C2-C3 bond is sandwiched between the carboxylate group of Glu367 and the pyrimidine ring of FAD, mechanism, overview
Desulfococcus multivorans
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
glutaryl-CoA + FAD
-
711251
Desulfococcus multivorans
glutaconyl-CoA + FADH2
-
-
-
?
additional information
the decarboxylating, EC 1.3.99.7, and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr to Val exchange strictly conserved between the two GDH types, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved Arg is stronger in GDHDes than in the decarboxylating enzyme, molecular dynamics. The identified structural changes prevent decarboxylation 1. by strengthening the C4-C5 bond of glutaconyl-CoA, 2. by reducing the leaving group potential of CO2, and 3. by increasing the distance between the C4 atom, negatively charged in the dienolate transition state, and the adjacent glutamic acid
711251
Desulfococcus multivorans
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
More
structure determination and comparison of human carboxylating GDH, EC 1.3.99.7, to the nondecarboxylating GDH, overview
Desulfococcus multivorans
tetramer
tetramer to monomer ration is 8.2:1, gel filtration
Desulfococcus multivorans
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Desulfococcus multivorans
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Desulfococcus multivorans
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
0.65 mol FAD/mol of enzyme, altered ratios of mutant enzymes, except for mutant V88S, overview. With the exception of V88S, all enzyme variants essentially loose the FAD cofactor
Desulfococcus multivorans
Cloned(Commentary) (protein specific)
Commentary
Organism
expression of His-tagged GDHDes in Escherichia coli
Desulfococcus multivorans
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
0.65 mol FAD/mol of enzyme, altered ratios of mutant enzymes, except for mutant V88S, overview. With the exception of V88S, all enzyme variants essentially loose the FAD cofactor
Desulfococcus multivorans
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified recombinant His-tagged GDHDes, hanging drop vapor diffusion method, 20 mg/ml protein in 10 mM MES, pH 6.0, 0.5 M KCl, 10% w/v glycerol, 1 mM DTT, 1 mM FAD, and 2 mM glutaryl-CoA are mixed with an equal volume of reservoir solution containing 50% v/v MPD, 0.1 M Tris-HCl, pH 8.5, and 0.2 M NH4H2PO4, at 4°C, X-ray diffraction structure determination and analysis
Desulfococcus multivorans
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
A80E
site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
A80E/S161T/V366Y
site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
A80E/V366Y
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
V366Y
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
V88S
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme. The mutant enzyme shows a higher tetramer conformation than the wild-type
Desulfococcus multivorans
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.014
-
glutaryl-CoA
pH 7.8, 30°C, mutant A80E/V366Y
Desulfococcus multivorans
0.028
-
glutaryl-CoA
pH 7.8, 30°C, mutant V366Y
Desulfococcus multivorans
0.053
-
glutaryl-CoA
pH 7.8, 30°C, wild-type enzyme
Desulfococcus multivorans
0.059
-
glutaryl-CoA
pH 7.8, 30°C, mutant V88S
Desulfococcus multivorans
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
glutaryl-CoA + FAD
Desulfococcus multivorans
-
glutaconyl-CoA + FADH2
-
-
?
additional information
Desulfococcus multivorans
the decarboxylating, EC 1.3.99.7, and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr to Val exchange strictly conserved between the two GDH types, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved Arg is stronger in GDHDes than in the decarboxylating enzyme, molecular dynamics. The identified structural changes prevent decarboxylation 1. by strengthening the C4-C5 bond of glutaconyl-CoA, 2. by reducing the leaving group potential of CO2, and 3. by increasing the distance between the C4 atom, negatively charged in the dienolate transition state, and the adjacent glutamic acid
?
-
-
-
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His-tagged GDHDes from Escherichia coli by nickel affinity chromatography
Desulfococcus multivorans
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
glutaryl-CoA + FAD
-
711251
Desulfococcus multivorans
glutaconyl-CoA + FADH2
-
-
-
?
additional information
the decarboxylating, EC 1.3.99.7, and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr to Val exchange strictly conserved between the two GDH types, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved Arg is stronger in GDHDes than in the decarboxylating enzyme, molecular dynamics. The identified structural changes prevent decarboxylation 1. by strengthening the C4-C5 bond of glutaconyl-CoA, 2. by reducing the leaving group potential of CO2, and 3. by increasing the distance between the C4 atom, negatively charged in the dienolate transition state, and the adjacent glutamic acid
711251
Desulfococcus multivorans
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
More
structure determination and comparison of human carboxylating GDH, EC 1.3.99.7, to the nondecarboxylating GDH, overview
Desulfococcus multivorans
tetramer
tetramer to monomer ration is 8.2:1, gel filtration
Desulfococcus multivorans
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Desulfococcus multivorans
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.8
-
assay at
Desulfococcus multivorans
General Information
General Information
Commentary
Organism
metabolism
the organism conserves the free energy of decarboxylation by a Na+-pumping glutaconyl-CoA decarboxylase. Glutaconyl-Co A-forming GDH is a nondecarboxylating enzyme
Desulfococcus multivorans
General Information (protein specific)
General Information
Commentary
Organism
metabolism
the organism conserves the free energy of decarboxylation by a Na+-pumping glutaconyl-CoA decarboxylase. Glutaconyl-Co A-forming GDH is a nondecarboxylating enzyme
Desulfococcus multivorans
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
10
-
glutaryl-CoA
pH 7.8, 30°C, mutants V366Y and A80E/V366Y
Desulfococcus multivorans
120
-
glutaryl-CoA
pH 7.8, 30°C, mutant V88S
Desulfococcus multivorans
1700
-
glutaryl-CoA
pH 7.8, 30°C, wild-type enzyme
Desulfococcus multivorans
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
10
-
glutaryl-CoA
pH 7.8, 30°C, mutants V366Y and A80E/V366Y
Desulfococcus multivorans
120
-
glutaryl-CoA
pH 7.8, 30°C, mutant V88S
Desulfococcus multivorans
1700
-
glutaryl-CoA
pH 7.8, 30°C, wild-type enzyme
Desulfococcus multivorans
Other publictions for EC 1.3.99.32
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)
711251
Wischgoll
Structural basis for promoting ...
Desulfococcus multivorans
Biochemistry
49
5350-5357
2010
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1
1
5
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4
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2
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1
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1
1
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2
2
1
-
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1
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1
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1
1
1
5
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4
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2
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1
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2
2
1
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1
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1
1
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3
3
712280
Wischgoll
Decarboxylating and nondecarbo ...
Desulfococcus multivorans
J. Bacteriol.
191
4401-4409
2009
-
-
1
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1
1
1
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2
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4
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1
-
-
-
-
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2
1
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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1
1
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2
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1
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2
1
1
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1
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