Cloned (Comment) | Organism |
---|---|
expression of His-tagged GDHDes in Escherichia coli | Desulfococcus multivorans |
Crystallization (Comment) | 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 |
Protein Variants | Comment | 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 Maximum [mM] | Substrate | Comment | 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Desulfococcus multivorans | C3UVB0 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged GDHDes from Escherichia coli by nickel affinity chromatography | Desulfococcus multivorans |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
glutaryl-CoA + FAD | - |
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 | Desulfococcus multivorans | ? | - |
? |
Subunits | Comment | 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 |
Synonyms | Comment | Organism |
---|---|---|
GDHDes | - |
Desulfococcus multivorans |
glutaryl-coenzyme A dehydrogenase | - |
Desulfococcus multivorans |
nondecarboxylating, glutaconyl-coenzyme A-forming GDH | - |
Desulfococcus multivorans |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Desulfococcus multivorans |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.8 | - |
assay at | Desulfococcus multivorans |
Cofactor | Comment | 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 |
General Information | Comment | 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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | 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 |