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.

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

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

Protein Variants

Protein Variants	Comment	Organism
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
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
E435Q	mutant protein is completely insoluble	Acetobacter aceti
E435Q	site-directed mutagenesis, the mutant is completely insoluble, 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
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
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

Inhibitors

Inhibitors	Comment	Organism
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	-

Reaction

Reaction	Comment	Organism	Reaction ID
succinyl-CoA + acetate = acetyl-CoA + succinate	the general half-reaction for class I CoA-transferases shows two tetrahedral oxyanion intermediates, which differ by whether CoA becomes attached to the external carbonyl, provided by the acyl-CoA/carboxylate substrate, or the internal carbonyl, provided by the essential active-site glutamate. Following exchange of the carboxylate product, the second half-reaction proceeds in the reverse order of the first half-reaction. In the first half-reaction, the binary enzyme·acyl-CoA complex is converted into a CoA thiolate complex that also contains an acylglutamyl anhydride adduct. Ping-pong kinetic mechanism. Val270 has a dual influence on carboxylate substrate selectivity, as a gate and as a clamp, Arg228 has an important kinetic role in carboxylate substrate binding. The auxiliary site nonselectively binds carboxylates at the threshold of the catalytic pocket, while selectivity is enforced by the conserved gating residue Val270 and the interior of the catalytic pocke	Acetobacter aceti	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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
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	?	-	?
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
AarC	-	Acetobacter aceti
acetic acid resistance factor	-	Acetobacter aceti
SCACT	-	Acetobacter aceti
SCOT	-	Acetobacter aceti
succinyl-CoA:3-oxoacid CoA-transferase	-	Acetobacter aceti
succinyl-CoA:acetate CoA-transferase	-	Acetobacter aceti
succinyl-coenzyme A:acetate CoA-transferase	-	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
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
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
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

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.000074	-	succinyl-CoA	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
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
0.0023	-	acetyl-CoA	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
0.005	-	acetyl-CoA	pH 8.0, 30°C, mutant E435D	Acetobacter aceti
0.009	-	succinyl-CoA	pH 8.0, 30°C, His6-tagged wild-type enzyme and 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
0.03	-	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
0.074	-	succinyl-CoA	mutant S71A, pH 8.0, temperature not specified in the publication	Acetobacter aceti
0.13	-	succinyl-CoA	mutant R228E, pH 8.0, temperature not specified in the publication	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
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
0.39	-	succinyl-CoA	mutant N347A, pH 8.0, temperature not specified in the publication	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
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
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
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
3.4	-	acetyl-CoA	wild-type, 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
5.2	-	acetyl-CoA	mutant E435D, pH 8.0, temperature not specified in the publication	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
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
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