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Literature summary extracted from

  • Wischgoll, S.; Demmer, U.; Warkentin, E.; Guenther, R.; Boll, M.; Ermler, U.
    Structural basis for promoting and preventing decarboxylation in glutaryl-coenzyme A dehydrogenases (2010), Biochemistry, 49, 5350-5357.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.99.32 expression of His-tagged GDHDes in Escherichia coli Desulfococcus multivorans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.3.8.6 purified GDH, hanging drop vapor diffusion method, 10 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 glutaryl-CoA, are mixed with an equal volume of reservoir solution containing 15% v/v MPD, 0.1 M imidazole, pH 6.5, 0.2 M KCl, and 2% PEG 3350, at 4°C, 2 days, X-ray diffraction structure determination and analysis Homo sapiens
1.3.99.32 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

EC Number Protein Variants Comment Organism
1.3.99.32 A80E site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type Desulfococcus multivorans
1.3.99.32 A80E/S161T/V366Y site-directed mutagenesis, inactive mutant. The mutant enzyme shows a higher tetramer conformation than the wild-type Desulfococcus multivorans
1.3.99.32 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
1.3.99.32 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
1.3.99.32 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.99.32 0.014
-
 glutaryl-CoA pH 7.8, 30°C, mutant A80E/V366Y Desulfococcus multivorans
1.3.99.32 0.028
-
 glutaryl-CoA pH 7.8, 30°C, mutant V366Y Desulfococcus multivorans
1.3.99.32 0.053
-
 glutaryl-CoA pH 7.8, 30°C, wild-type enzyme Desulfococcus multivorans
1.3.99.32 0.059
-
 glutaryl-CoA pH 7.8, 30°C, mutant V88S Desulfococcus multivorans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.8.6 glutaryl-CoA + FAD Homo sapiens
-
 crotonoyl-CoA + CO2 + FADH2
-
 ?
1.3.8.6 additional information Homo sapiens the decarboxylating and nondecarboxylating, EC 1.3.99.X from Desulfococcus multivorans, 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 ?
-
 ?
1.3.99.32 glutaryl-CoA + FAD Desulfococcus multivorans
-
 glutaconyl-CoA + FADH2
-
 ?
1.3.99.32 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

EC Number Organism UniProt Comment Textmining
1.3.8.6 Homo sapiens Q92947
-
-
1.3.99.32 Desulfococcus multivorans C3UVB0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.99.32 recombinant His-tagged GDHDes from Escherichia coli by nickel affinity chromatography Desulfococcus multivorans

Reaction

EC Number Reaction Comment Organism Reaction ID
1.3.99.32 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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.8.6 glutaryl-CoA + FAD
-
 Homo sapiens crotonoyl-CoA + CO2 + FADH2
-
 ?
1.3.8.6 glutaryl-CoA + FAD detection using ferrocenium hexafluorophosphate Homo sapiens crotonoyl-CoA + CO2 + FADH2
-
 ?
1.3.8.6 additional information the decarboxylating and nondecarboxylating, EC 1.3.99.X from Desulfococcus multivorans, 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 Homo sapiens ?
-
 ?
1.3.99.32 glutaryl-CoA + FAD
-
 Desulfococcus multivorans glutaconyl-CoA + FADH2
-
 ?
1.3.99.32 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

EC Number Subunits Comment Organism
1.3.8.6 More structure determination and comparison of carboxylating GDH to the Desulfococcus multivorans nondecarboxylating GDH, EC 1.3.99.X, overview Homo sapiens
1.3.99.32 More structure determination and comparison of human carboxylating GDH, EC 1.3.99.7, to the nondecarboxylating GDH, overview Desulfococcus multivorans
1.3.99.32 tetramer tetramer to monomer ration is 8.2:1, gel filtration Desulfococcus multivorans

Synonyms

EC Number Synonyms Comment Organism
1.3.8.6 GDH
-
 Homo sapiens
1.3.8.6 glutaryl-coenzyme A dehydrogenase
-
 Homo sapiens
1.3.99.32 GDHDes
-
 Desulfococcus multivorans
1.3.99.32 glutaryl-coenzyme A dehydrogenase
-
 Desulfococcus multivorans
1.3.99.32 nondecarboxylating, glutaconyl-coenzyme A-forming GDH
-
 Desulfococcus multivorans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.3.8.6 30
-
 assay at Homo sapiens
1.3.99.32 30
-
 assay at Desulfococcus multivorans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.3.8.6 7.8
-
 assay at Homo sapiens
1.3.99.32 7.8
-
 assay at Desulfococcus multivorans

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.8.6 FAD
-
 Homo sapiens
1.3.99.32 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

EC Number General Information Comment Organism
1.3.8.6 metabolism glutaryl-coenzyme A dehydrogenases involved in amino acid degradation catalyze both the dehydrogenation and decarboxylation of glutaryl-CoA to crotonoyl-CoA and CO2 Homo sapiens
1.3.99.32 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.3.99.32 10
-
 glutaryl-CoA pH 7.8, 30°C, mutants V366Y and A80E/V366Y Desulfococcus multivorans
1.3.99.32 120
-
 glutaryl-CoA pH 7.8, 30°C, mutant V88S Desulfococcus multivorans
1.3.99.32 1700
-
 glutaryl-CoA pH 7.8, 30°C, wild-type enzyme Desulfococcus multivorans