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Information on EC 2.3.3.16 - citrate synthase (unknown stereospecificity) and Organism(s) Escherichia coli and UniProt Accession P0ABH7
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2.3.3.16 citrate synthase (unknown stereospecificity)IUBMB Comments
This entry has been included to accommodate those citrate synthases for which the stereospecificity with respect to C-2 of oxaloacetate has not been established [cf. EC 2.3.3.1, citrate (Si)-synthase and EC 2.3.3.3, citrate (Re)-synthase].
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
citrate synthetase, mitochondrial citrate synthase, peroxisomal citrate synthase, si-citrate synthase, type ii citrate synthase, citrate condensing enzyme, citrate synthase cit1, bifunctional citrate synthase/2-methylcitrate synthase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
citrate condensing enzyme
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citrate oxaloacetate-lyase (CoA-acetylating)
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citrate oxaloacetate-lyase, CoA-acetylating
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citrate synthase
citrate synthetase
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citric synthase
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citric-condensing enzyme
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-
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citrogenase
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oxalacetic transacetase
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oxaloacetate transacetase
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synthase, citrate
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citrate synthase
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-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
PATHWAY SOURCE
PATHWAYS
-
-, -, -, -, -, -, -, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:oxaloacetate C-acetyltransferase (thioester-hydrolysing)
This entry has been included to accommodate those citrate synthases for which the stereospecificity with respect to C-2 of oxaloacetate has not been established [cf. EC 2.3.3.1, citrate (Si)-synthase and EC 2.3.3.3, citrate (Re)-synthase].
CAS REGISTRY NUMBER
COMMENTARY
9027-96-7
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
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mechanism for domain closure. It appears that there is a common barrier between the open- and closed-domain conformations that cannot be overcome in either exploring or targeting simulations. For citrate synthase in the open conformation there are 258 atoms from both domains that are in the domain-contact sets. This increases to only 284 for the closed structure. These atoms come from 61 residues in the open which increases to 66 in the closed. Of these, 57 are common to both open and closed conformations
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
KCl
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADH
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inhibition of wild-type enzyme. Inhibition is extremenly weak in mutant enzymes Y145A, R163L, and K167A
NADH
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0.1 M, 92% inhibition of wild-type, 25% inhibition of K283 acetylated variant, 88% inhibition of K295 acetylated variant, 7% inhibition of K168 acetylated variant
additional information
allosteric inhibition mechanism, structure relationship
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additional information
-
allosteric inhibition mechanism, structure relationship
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
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mutants, with and without KCl, overview
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0.69
acetyl-CoA
mutant enzyme G181E, pH and temperature not specified in the publication
0.75
acetyl-CoA
wild type enzyme, pH and temperature not specified in the publication
1.01
acetyl-CoA
mutant enzyme T204R, pH and temperature not specified in the publication
0.0105
oxaloacetate
mutant enzyme G181E, pH and temperature not specified in the publication
0.011
oxaloacetate
wild type enzyme, pH and temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
chimeric mutants, overview
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17
acetyl-CoA
mutant enzyme T204R, pH and temperature not specified in the publication
18
acetyl-CoA
mutant enzyme G181E, pH and temperature not specified in the publication
44
acetyl-CoA
wild type enzyme, pH and temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
120
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purified recombinant chimeric E. coli-type protein with small Acinetobacter domain
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
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recombinant wild-type CS has no detectable acetylation. Acetylation of lysine residues does not result in significantly different activities with that of the wild-type, except for residues K283 and K295. Acetylation at K283 increases the activity by nearly twofold, while acetylation at K295 decreased the activity by about 10fold. CS can be acetylated by acetyl-phosphate chemically, and be deacetylated by the CobB deacetylase
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexamer
additional information
additional information
subunit organization and crystal structure, amino acid residues involved in subunit interaction
additional information
-
subunit organization and crystal structure, amino acid residues involved in subunit interaction
additional information
-
recombinant chimeric enzymes, domain functions, subunit organisation
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method, from 2-2.3 M ammonium sulfate, 2% v/v polyethylene glycol 400, 0.1 M HEPS, pH 6.0, X-ray diffraction analysis, structure determination and modeling: 3 identical dimer units arranged about a central 3-fold axis
hanging-drop vapour-diffusion method from 2.0-2.2 M ammonium sulfate, 2% PEG 400, and 0.1 M Na-HEPES at pH 6.0. the NADH-bound form of mutant R109L is obtained by soaking a variant crystal in a solution containing 1.22 mM NADH, 2.8 M ammonium sulfate, 2% polyethylene glycol 400 and 0.1 M Na-Hepes at pH 6.0
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
G181E
the mutant shows reduced activity compared to the wild type enzyme and is not inhibited by NADH
T204R
the mutant shows reduced activity compared to the wild type enzyme and is not inhibited by NADH
D362A
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acetyl-CoA binding site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate
H229Q
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active site mutant, reduced turnover, increased Ki for 2-oxoglutarate
H264A
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acetyl-CoA binding site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate
K167A
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extremely weak inhibition by NADH. Does not form hexamers in response to NADH, unlike the wild-type enzyme
R109L
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extremely weak inhibition by NADH. Great structural change. Both regions - residue 260-311 and 316-342 - are much less mobile than in wild-type enzyme
R163L
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extremely weak inhibition by NADH. Does not form hexamers in response to NADH, unlike the wild-type enzyme
R407L
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active site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate
additional information
additional information
-
effect of active-site mutantions on substrate binding
additional information
-
knockout of citrate syntase gltA or phosphoenolpyruvate carboxylase ppc decreases the maximum cell density by 10% and 7%, resp. Over-expression of gltA or ppc increases the maximum cell dry weight by 23% and 91% resp. No acetate excretion is detected at these increased cell densities
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of a chimeric protein with one Acinetobacter domain in Escherichia coli, domain interactions, subunit interactions
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
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over-expression of citrate syntase gltA or phosphoenolpyruvate carboxylase ppc increases the maximum cell dry weight by 23% and 91% resp. No acetate excretion is detected at these increased cell densities
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Robinson, M.S.; Easom, R.A.; Danson, M.J.; Weitzman, P.D.J.
Citrate synthase of Escherichia coli. Characterization of the enzyme from a plasmid-cloned gene and amplification of the intracellular levels
FEBS Lett.
154
51-54
1983
Escherichia coli
Pereira, D.S.; Donald, L.J.; Hesfield, D.J.; Duckworth, H.W.
Active site mutants of Escherichia coli citrate synthase
J. Biol. Chem.
269
412-417
1994
Escherichia coli
Molgat, G.F.; Donald, L.J.; Duckworth, H.W.
Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes
Arch. Biochem. Biophys.
298
238-246
1992
Acinetobacter calcoaceticus subsp. anitratus, Escherichia coli
Nguyen, N.T.; Maurus, R.; Stokell, D.J.; Ayed, A.; Duckworth, H.W.; Brayer, G.D.
Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes
Biochemistry
40
13177-13187
2001
Escherichia coli (P0ABH7), Escherichia coli
Stokell, D.J.; Donald, L.J.; Maurus, R.; Nguyen, N.T.; Sadler, G.; Choudhary, K.; Hultin, P.G.; Brayer, G.D.; Duckworth, H.W.
Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli
J. Biol. Chem.
278
35435-35443
2003
Escherichia coli
De Maeseneire, S.L.; De Mey, M.; Vandedrinck, S.; Vandamme, E.J.
Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures
Biotechnol. Lett.
28
1945-1953
2006
Escherichia coli
Snow, C.; Qi, G.; Hayward, S.
Essential dynamics sampling study of adenylate kinase: comparison to citrate synthase and implication for the hinge and shear mechanisms of domain motions
Proteins
67
325-337
2007
Escherichia coli
Buch, A.D.; Archana, G.; Kumar, G.N.
Enhanced citric acid biosynthesis in Pseudomonas fluorescens ATCC 13525 by overexpression of the Escherichia coli citrate synthase gene
Microbiology
155
2620-2629
2009
Escherichia coli
Duckworth, H.W.; Nguyen, N.T.; Gao, Y.; Donald, L.J.; Maurus, R.; Ayed, A.; Bruneau, B.; Brayer, G.D.
Enzyme-substrate complexes of allosteric citrate synthase: evidence for a novel intermediate in substrate binding
Biochim. Biophys. Acta
1834
2546-2553
2013
Escherichia coli (P0ABH7)
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