Literature summary for 2.8.3.18 extracted from

  • Mullins, E.A.; Kappock, T.J.
    Crystal structures of Acetobacter aceti succinyl-coenzyme A (CoA):acetate CoA-transferase reveal specificity determinants and illustrate the mechanism used by class I CoA-transferases (2012), Biochemistry, 51, 8422-8434.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Acetobacter aceti
gene aarC, sequence comparisons and phylogenetic analysis, expression of C-terminally His6-tagged wild-type and mutant enzymes Acetobacter aceti

Crystallization (Commentary)

Crystallization (Comment) Organism
crystal structures of a C-terminally His6-tagged form of several wild-type and mutant complexes, including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. Mutant E294A crystallizes in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2'' CoA is nearly immobile along its entire length during all stages of the enzyme reaction Acetobacter aceti
native and C-terminally His6-tagged wild-type enzymes in complexes including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts, hanging drop vapor diffusion method, mixing of 0.002 ml of protein solution containing 5.6 mg/ml AarC in 45 mM potassium phosphate, pH 8.0, 90 mM potassium chloride, and 2 mM CoA or 6.0 mg/ml His6-tagged AarC in 45 mM Tris-HCl, pH 8.0, 90 mM potassium chloride, and 2 mM CoA, with 0.002 ml of reservoir solution containing 0.8-1.0 M sodium citrate, 0.1 M imidazole, pH 8.2, and 25 mM 2-mercaptoethanol for orthorhombic crystals or 1.7-2.0 M ammonium sulfate, 0.2 M sodium chloride, 0.1 M sodium cacodylate, pH 6.5, and 25 mM 2-mercaptoethanol for hexagonal crystals, room temperature of about 22°C, X-ray diffraction structure determination and analysis Acetobacter aceti

Engineering

Protein Variants Comment Organism
S71A large decrease in catalytic activity Acetobacter aceti
S71A site-directed mutagenesis, the mutant shows impaired catalytic activity associated with lower kcat values, ligand bound crystal structure modeling Acetobacter aceti
E294A complete loss of activity Acetobacter aceti
E294A site-directed mutagenesis, the mutant specific catalytic activity is 10000fold reduced compared to the wild-type enzyme, ligand bound crystal structure modeling Acetobacter aceti
E435A mutant protein is completely insoluble Acetobacter aceti
E435A site-directed mutagenesis, the mutant is completely insoluble, ligand bound crystal structure determination and analysis, the mutant crystallizes in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation Acetobacter aceti
E435Q mutant protein is completely insoluble Acetobacter aceti
E435Q site-directed mutagenesis, the mutant is completely insoluble, ligand bound crystal structure modeling Acetobacter aceti
R228E large decrease in catalytic activity Acetobacter aceti
R228E site-directed mutagenesis, the mutant has a specific defect in its ability to bind both carboxylate substrates, ligand bound crystal structure modeling Acetobacter aceti
N347A large decrease in catalytic activity Acetobacter aceti
N347A site-directed mutagenesis, the mutant shows impaired catalytic activity, but the apparent affinities for all four substrates are largely unaffected, ligand bound crystal structure modeling Acetobacter aceti
E435D activity similar to wild-type Acetobacter aceti
E435D site-directed mutagenesis, the mutant catalytic properties are nearly equivalent to those of the His6-tagged wild-type enzyme, ligand bound crystal structure modeling Acetobacter aceti

Inhibitors

Inhibitors Comment Organism Structure
acetate
-
Acetobacter aceti
citrate weak competitive inhibition against succinate Acetobacter aceti
succinate weak competitive inhibition Acetobacter aceti

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetic analysis using a Michaelis-Menten model, a substrate inhibition model, or a competitive inhibition model, overview. Arg228 has an important kinetic role in carboxylate substrate binding Acetobacter aceti

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
succinyl-CoA + acetate Acetobacter aceti via an acetylglutamyl anhydride intermediate and glutamyl-CoA thioester adduct acetyl-CoA + succinate
-
?

Organism

Organism UniProt Comment Textmining
Acetobacter aceti B3EY95
-
-
Acetobacter aceti B3EY95 gene aarC
-
Acetobacter aceti 1023 B3EY95 gene aarC
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information no activity with glycolate, glyoxylate, oxalate, trifluoroacetate, DL-lactate, L-lactate, malonate, pyruvate, maleate, butyrate, D-tartrate, L-tartrate, 2-oxooglutarate, citrate, or DL-isocitrate Acetobacter aceti ?
-
?
additional information substrate specificity, overview. Acetoacetate, propionate, D-malate, fumarate, L-malate, formate, oxaloacetate, DL-methylsuccinate, glutarate are alternate substrates, no activity with glycolate, glyoxylate, oxalate, trifluoroacetate, DL-lactate, L-lactate, malonate, pyruvate, maleate, butyrate, D-tartrate, L-tartrate, 2-oxooglutarate, citrate, or DL-isocitrate Acetobacter aceti ?
-
?
additional information no activity with glycolate, glyoxylate, oxalate, trifluoroacetate, DL-lactate, L-lactate, malonate, pyruvate, maleate, butyrate, D-tartrate, L-tartrate, 2-oxooglutarate, citrate, or DL-isocitrate Acetobacter aceti 1023 ?
-
?
additional information substrate specificity, overview. Acetoacetate, propionate, D-malate, fumarate, L-malate, formate, oxaloacetate, DL-methylsuccinate, glutarate are alternate substrates, no activity with glycolate, glyoxylate, oxalate, trifluoroacetate, DL-lactate, L-lactate, malonate, pyruvate, maleate, butyrate, D-tartrate, L-tartrate, 2-oxooglutarate, citrate, or DL-isocitrate Acetobacter aceti 1023 ?
-
?
acetyl-CoA + acetoacetate 0.35% of the activity with succinate Acetobacter aceti acetoacetyl-CoA + acetate
-
r
acetyl-CoA + D-malate 0.28% of the activity with succinate Acetobacter aceti D-malyl-CoA + acetate
-
r
acetyl-CoA + fumarate 0.20% of the activity with succinate Acetobacter aceti fumaryl-CoA + acetate
-
r
acetyl-CoA + propionate 0.34% of the activity with succinate Acetobacter aceti propionyl-CoA + acetate
-
r
acetyl-CoA + succinate
-
Acetobacter aceti succinyl-CoA + acetate
-
r
succinyl-CoA + acetate 5.1% of the activity with succinate + acetyl-CoA Acetobacter aceti acetyl-CoA + succinate
-
r
succinyl-CoA + acetate via an acetylglutamyl anhydride intermediate and glutamyl-CoA thioester adduct Acetobacter aceti acetyl-CoA + succinate
-
?
succinyl-CoA + acetoacetate
-
Acetobacter aceti acetoacetyl-CoA + succinate
-
?
succinyl-CoA + D-malate
-
Acetobacter aceti D-malyl-CoA + succinate
-
?
succinyl-CoA + DL-methylsuccinate
-
Acetobacter aceti DL-methylsuccinyl-CoA + succinate
-
?
succinyl-CoA + formate
-
Acetobacter aceti formyl-CoA + succinate
-
?
succinyl-CoA + fumarate
-
Acetobacter aceti fumaryl-CoA + succinate
-
?
succinyl-CoA + glutarate
-
Acetobacter aceti glutaryl-CoA + succinate
-
?
succinyl-CoA + L-malate
-
Acetobacter aceti L-malyl-CoA + succinate
-
?
succinyl-CoA + oxaloacetate
-
Acetobacter aceti oxaloacetyl-CoA + succinate
-
?
succinyl-CoA + propionate
-
Acetobacter aceti propionyl-CoA + succinate
-
?

Synonyms

Synonyms Comment Organism
SCOT
-
Acetobacter aceti
succinyl-CoA:3-oxoacid CoA-transferase
-
Acetobacter aceti
succinyl-CoA:acetate CoA-transferase
-
Acetobacter aceti
SCACT
-
Acetobacter aceti
succinyl-coenzyme A:acetate CoA-transferase
-
Acetobacter aceti
AarC
-
Acetobacter aceti
acetic acid resistance factor
-
Acetobacter aceti

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Acetobacter aceti

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Acetobacter aceti

pH Stability

pH Stability pH Stability Maximum Comment Organism
3.8
-
unstable below Acetobacter aceti
3.8 6.8
-
Acetobacter aceti

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
150
-
succinate pH 8.0, 30°C Acetobacter aceti
150
-
citrate pH 8.0, temperature not specified in the publication Acetobacter aceti
1600
-
acetate pH 8.0, temperature not specified in the publication Acetobacter aceti

General Information

General Information Comment Organism
physiological function the enzyme is an acetic acid resistance factor AarC that is required for acetate resistance by vinegar factory strain Acetobacter aceti 1023. The enzyme acts in a variant citric acid cycle that overoxidizes acetic acid to CO2, which then diffuses into the acidic culture medium Acetobacter aceti
evolution the enzyme belongs to the class I-CoA-transferases, which, typified by mitochondrial succinyl-CoA:3-oxoacid CoA-transferase, form multiple covalent adducts involving an essential glutamate residue. Arg228 is found in only AarC and several closely allied SCACT group sequences, EC 6.2.1 Acetobacter aceti
additional information the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2''. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases, structural model for the AarC mechanism, overview. An auxiliary carboxylate binding site, located just outside the AarC catalytic pocket, contributes to the efficient recognition and conversion of the physiological carboxylate substrates. Protein conformational dynamics, overview. Arg228 has an important kinetic role in carboxylate substrate binding. Regulation of carboxylate access to the active-site glutamate, overview Acetobacter aceti

KCat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.7
-
acetyl-CoA mutant N347A, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.7
-
acetyl-CoA mutant S71A, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.03
-
succinate mutant S71A, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.03
-
succinate pH 8.0, 30°C, mutant S71A Acetobacter aceti
0.16
-
acetate mutant N347A, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.16
-
acetate pH 8.0, 30°C, mutant N347A Acetobacter aceti
9
-
succinyl-CoA wild-type, pH 8.0, temperature not specified in the publication Acetobacter aceti
9
-
succinyl-CoA mutant E435D, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.3
-
succinate mutant R228E, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.3
-
succinate pH 8.0, 30°C, mutant R228E Acetobacter aceti
0.0034
-
acetyl-CoA pH 8.0, 30°C, His6-tagged wild-type enzyme Acetobacter aceti
0.13
-
succinyl-CoA mutant R228E, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.074
-
succinyl-CoA mutant S71A, pH 8.0, temperature not specified in the publication Acetobacter aceti
4
-
acetate wild-type, pH 8.0, temperature not specified in the publication Acetobacter aceti
4
-
acetate pH 8.0, 30°C, His6-tagged wild-type enzyme Acetobacter aceti
0.0023
-
acetyl-CoA pH 8.0, 30°C, mutant R228E Acetobacter aceti
5.2
-
acetyl-CoA mutant E435D, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.005
-
acetyl-CoA pH 8.0, 30°C, mutant E435D Acetobacter aceti
0.0007
-
acetyl-CoA pH 8.0, 30°C, mutant N347A Acetobacter aceti
0.0007
-
acetyl-CoA pH 8.0, 30°C, mutant S71A Acetobacter aceti
0.009
-
succinyl-CoA pH 8.0, 30°C, His6-tagged wild-type enzyme and mutant E435D Acetobacter aceti
3.4
-
acetyl-CoA wild-type, pH 8.0, temperature not specified in the publication Acetobacter aceti
79
-
succinate wild-type, pH 8.0, temperature not specified in the publication Acetobacter aceti
79
-
succinate pH 8.0, 30°C, His6-tagged wild-type enzyme Acetobacter aceti
0.39
-
succinyl-CoA mutant N347A, pH 8.0, temperature not specified in the publication Acetobacter aceti
3
-
acetate mutant E435D, pH 8.0, temperature not specified in the publication Acetobacter aceti
3
-
acetate pH 8.0, 30°C, mutant E435D Acetobacter aceti
0.6
-
acetate mutant R228E, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.6
-
acetate pH 8.0, 30°C, mutant R228E Acetobacter aceti
2.7
-
succinate mutant N347A, pH 8.0, temperature not specified in the publication Acetobacter aceti
2.7
-
succinate pH 8.0, 30°C, mutant N347A Acetobacter aceti
0.000074
-
succinyl-CoA pH 8.0, 30°C, mutant S71A Acetobacter aceti
73
-
succinate mutant E435D, pH 8.0, temperature not specified in the publication Acetobacter aceti
73
-
succinate pH 8.0, 30°C, mutant E435D Acetobacter aceti
0.029
-
acetate mutant S71A, pH 8.0, temperature not specified in the publication Acetobacter aceti
0.029
-
acetate pH 8.0, 30°C, mutant S71A Acetobacter aceti
0.00013
-
succinyl-CoA pH 8.0, 30°C, mutant R228E Acetobacter aceti
0.00039
-
succinyl-CoA pH 8.0, 30°C, mutant N347A Acetobacter aceti