Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.3.99.32 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)

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

Crystallization (Commentary)

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Desulfococcus multivorans C3UVB0
-
-

Purification (Commentary)

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

Reaction

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 and Products (Substrate)

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

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

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 [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Desulfococcus multivorans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
assay at Desulfococcus multivorans

Cofactor

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

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 [mM/s]

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