Information on EC 2.3.3.16 - citrate synthase (unknown stereospecificity)

oxaloacetate + acetyl-CoA + H2O

citrate + CoA

Substrates: -
Products: -

?

propanoyl-CoA + H2O + oxaloacetate

(2S,3S)-2-hydroxybutane-1,2,3-tricarboxylate + CoA

propionyl-CoA + H2O + oxaloacetate

(2S,3S)-2-methylcitrate + CoA

propionyl-CoA + oxaloacetate + H2O

2-methylcitrate + CoA

additional information

?

-

12 entries

acetyl-CoA + H2O + oxaloacetate

citrate + CoA

37 entries

acetyl-CoA + oxaloacetate + H2O

citrate + CoA

33 entries

propanoyl-CoA + H2O + oxaloacetate

(2S,3S)-2-hydroxybutane-1,2,3-tricarboxylate + CoA

propionyl-CoA + H2O + oxaloacetate

(2S,3S)-2-methylcitrate + CoA

additional information

?

-

AMP

(NH4)2SO4

-

activates at at least 0.5 mM

Ca2+

-

as CaCl2, inhibition at concentrations higher than 50 mM

K+

KCl

Mg2+

Na+

112.6% activity at 2 mM

NaCl

phosphate

-

additional information

2-oxoglutarate

18 entries

5,5'-dithiobis(2-nitrobenzoate)

abemaciclib

-

competitive inhibition with respect to acetyl-CoA, uncompetitive inhibition with respect to oxaloacetate

-

acetyl-CoA

ACMX

-

competitive inhibitor versus acetyl-CoA

ADP

Ag+

almost complete inhibition at 2 mM

AMP

ATP

bazedoxifene

-

competitive inhibition with respect to acetyl-CoA, uncompetitive inhibition with respect to oxaloacetate

Ca2+

-

CaCl2

cAMP

-

no inhibition

Cations

-

monovalent and divalent

-

citrate

Co2+

CoA

CTP

-

poor

Cu2+

dethiaacetyl-CoA

partial substrate

Dithionitrobenzoate

-

dithiothreitol

Q9R6Y4

2 mM, almost complete loss of activity

EDTA

Q9R6Y4

2 mM, 59% residual activity

elongation factor 1alpha

Tetrahymena pyriformis

-

causes polymerization of 49K protein, reduced activity

488064

-

GTP

-

poor

Guanidinium chloride

-

irreversible inactivation of recombinant wild-type at 1.6 M and of recombinant mutant G196V at 0.2 M, at 0.5 M activation of the wild-type

H2O2

about 54% inhibition at 0.4 mM

Hg2+

95% inhibition at 2 mM

HgCl2

imatinib

-

competitive inhibition with respect to acetyl-CoA, uncompetitive inhibition with respect to oxaloacetate

iodoacetamide

KCl

L-malate

-

no inhibition

lauroyl-CoA

-

Mg2+

MgCl2

Mn2+

MnCl2

-

56% inhibition at 10 mM

N-ethylmaleimide

-

strong

NaCl

NAD+

-

10 mM, 9% inhibition

NADH

NADP+

NADPH

Ni2+

oxaloacetate

p-chloromercuribenzoate

p-hydroxymercuribenzoate

-

60% inhibition at 0.1 mM, protection by oxaloacetate

488039

Pb2+

almost complete inhibition at 2 mM

phosphoenolpyruvate

5 mM, 14% residual activity

propionyl-CoA

-

competitive against acetyl-CoA

S-carboxymethyl-CoA

competitive inhibition versus acetyl-CoA, non-competitive inhibition versus oxaloacetate; inhibits the native enzyme competitively versus acetyl-CoA and non-competitively versus oxaloacetate

724900

SDS

-

strong inhibition

succinyl-CoA

-

mixed-type inhibition

TTP

-

poor

Urea

-

irreversible inactivation of recombinant wild-type at 9.3 M and of recombinant mutant G196V at 5 M, at up to 8 M activation of the wild-type

UTP

-

poor

vorapaxar

-

competitive inhibition with respect to acetyl-CoA, uncompetitive inhibition with respect to oxaloacetate

-

Zn2+

additional information

23 entries

-

acetate

-

alpha-ketoglutarate

-

ammonium sulfate

-

activates at 0.5 mM

AMP

CaCl2

100 mM, 151% increase in activity

Guanidinium chloride

-

activation of recombinant wild-type 1.6fold at 0.5 M, inactivation at 1.6 M

KCl

100 mM, 88% increase in activity. Activation is pH dependent

light

-

light activates up to 1.4fold, regulation in correlation with photosynthesis

488053

-

MgCl2

100 mM, 291% increase in activity

NaCl

100 mM, 105% increase in activity

thioredoxin

a strong and more than 8fold activation of enzyme activity is observed for isoform CS4 with Escherichia coli thioredoxin coupled to reduction by NTR and NADPH

Urea

0.00067 - 125

acetyl-CoA

107 entries

0.0005 - 0.001

citrate

0.00114 - 90.3

oxaloacetate

99 entries

0.0017 - 0.016

propionyl-CoA

additional information

-

0.0001 - 262.8

acetyl-CoA

51 entries

0.5 - 2100

citrate

9 entries

0.0001 - 230.7

oxaloacetate

30 entries

4 - 12

propionyl-CoA

additional information

8 entries

-

7.4 - 4500

acetyl-CoA

9 entries

14.2 - 20700

oxaloacetate

9 entries

0.3 - 20

2-oxoglutarate

0.00211 - 0.0029

abemaciclib

-

0.125 - 30

ATP

21 entries

0.00217 - 0.00325

bazedoxifene

2.4 - 7.19

citrate

0.11

CoA

-

0.00215 - 0.00298

imatinib

0.0017 - 12

NADH

27 entries

0.8 - 4.9

NADPH

0.003 - 0.051

oxaloacetate

0.0024 - 0.0225

propionyl-CoA

0.00037 - 0.0024

S-carboxymethyl-CoA

0.00278 - 0.00361

vorapaxar

-

additional information

-

mutants, overview

-

0.00023 - 0.00028

-

wild-type

0.00078

Daucus carota

O80433

recombinant in transgenic Arabidopsis thaliana plants

0.002

Methanosarcina barkeri

-

0.008

-

recombinant purified mutant G196V

Natronobacterium gregoryi

0.014

-

0.02

Solanum tuberosum

-

recombinant enzyme in crude E. coli enzyme extract

488054

0.026

Natronomonas pharaonis

-

0.045

-

crude extract

0.051

-

0.061

-

0.083

-

recombinant purified wild-type

0.172

-

0.23 - 0.28

-

0.28

-

0.331

-

crude enzyme from torpid skeletal muscle, at pH 8.0 and 22°C

0.3844

-

crude enzyme from euthermic skeletal muscle, at pH 8.0 and 22°C

0.76

-

isoenzyme CSI, Matrex Gel Red-A

0.764

Citrullus lanatus

-

purified glyoxysomal isozyme

488047

0.78

Daucus carota

O80433

transgenic Arabidopsis thaliana plants overexpressing the Daucus carota citrate synthase

0.87

Cicer arietinum

-

partially purified enzyme

0.99

-

glucose-grown cells, pH and temperature not specified in the publication

1.25

-

partially purified enzyme

1.26

-

acetate-grown cells, pH and temperature not specified in the publication

1.32

-

isoenzymes CSI and CSII, DEAE-Sephacel

102.7

-

purified enzyme

11.5

-

isoenzyme CSII, Matrex Gel Red-A

112.5

1190

-

partially purified enzyme

120

-

purified recombinant chimeric E. coli-type protein with small Acinetobacter domain

14

-

partially purified enzyme

18

-

purified enzyme

488048

18.8

-

partially purified enzyme

2.6

-

partially purified enzyme

21.6

213

-

purified fusion protein of citrate synthase and mitochondrial malate dehydrogenase, coupled assay

488077

22.545

-

68.1fold purified enzyme from torpid skeletal muscle, at pH 8.0 and 22°C

230

-

purified enzyme

235

-

purified enzyme

24

-

purified enzyme

25.8

-

purified enzyme

488066

27.45

-

488033

28

Rhodothermus marinus

-

purified dimeric and hexameric enzyme form

488087

288

-

purified enzyme

30

-

purified recombinant enzyme from E. coli

31.714

-

82.5fold purified enzyme from euthermic skeletal muscle, at pH 8.0 and 22°C

35.1

Tetrahymena pyriformis

-

purified enzyme

488063

4.7

-

isozymes CS I and CS II

4.71

-

isoenzyme CSI, Sephadex G-200

40

37°C, pH 8.0

41

-

41.5

Q9TEM3

citrate synthase activity of a purified methylcitrate synthase

488089

45

-

purified wild-type enzyme

471.7

-

partially purified enzyme

50 - 60

-

partially purified enzyme

51.4

-

wild-type

52.3

-

purified enzyme

523

-

53

-

57

-

purified recombinant chimeric Acinetobacter-type protein with small E. coli domain

60

Rhodothermus marinus

-

dimeric and hexameric enzyme

488087

62.6

-

purified enzyme

64

-

purified enzyme

488050

664.2

-

488053

70

-

purified recombinant isozyme CS II

79

purified enzyme

88

-

purified wild-type enzyme

9

-

S43C mutant

additional information

5 - 6

-

7

-

7.3 - 7.8

-

7.4

-

enzyme assay at

7.5

7.5 - 8

7.6

7.8

8

8 - 9

-

8.1

8.2

-

8.5

8.6

-

8.6 - 9

-

9

Q9R6Y4

-

additional information

5 - 9

6 - 10

Geobacter sulfurreducens

Q6JGI2

about 30% of maximal activity at pH 6 and at pH 10

657610

7 - 9

Agave americana

-

488038

7.2 - 9

-

20

23

-

enzyme assay at

25

30

34

-

enzyme assay at

35

37

40

-

45

-

55

65

-

enzyme mutant lacking 13 amino acid residues of the C-terminal

65 - 70

70

-

488082

70 - 75

-

wild-type

75

80

85

-

dimeric and hexameric enzyme forms

90

20 - 50

-

35

nearly 50% of the activity is retained at 35°C

40 - 90

6.46

Geobacter sulfurreducens

Q6JGI2

calculation from nucleotide sequence

6.66

calculated

6.7

7.8

-

calculated from amino acid sequence

8.1

Fasciola hepatica

M4QLE7

calculated from amino acid sequence

736942

8.5

Homo sapiens

O75390

calculated from amino acid sequence

736278

8.6

8.8

6 entries

8.9

wild-type protein, isoelectric focusing

9.1

9.5

F4ZC76

calculated from sequence

757999

Acetobacter aceti

hexameric type II citrate synthase

672091

P20901

Uniprot

Agrobacterium tumefaciens

-

488030, 488069

-

Agave americana

-

488038

-

-

674243

-

Agrobacterium tumefaciens C58 / ATCC 33970

-

674243

-

Amblyrhynchus cristatus

-

686223

Q0GNE1

SwissProt

Aquipseudomonas alcaligenes

-

-

Aquipseudomonas alcaligenes D 123

-

-

Arabidopsis thaliana WS

-

-

-

-

-

-

-

-

Aspergillus nidulans FGSC A26

formerly Aspergillus nidulans

UniProt

Aspergillus niger ANJ-120

-

757903

T1SGS1

UniProt

Aspergillus niger N400 / DSM 2466 / ATCC 9029 / CBS 120.49

gene citA

488074

Q9P8D6

SwissProt

Azotobacter vinelandii OP

-

488028, 488030

-

-

-

Bacillus subtilis 168

-

747131

P45858

SwissProt

Bacillus subtilis HS2A2

-

-

Bacillus subtilis S2A1

-

488044

-

Brachypodium distachyon

aluminium-tolerant line ABR8 and aluminium-sensitive line ABR1

777498

-

Brassica napus

cultivars Zhongshuang 9, H305, Qing 8, 9558, H04, H1020, PH36, Huyou 17, GH01, Zhongshuang 6, 2021, and Zhongshuang 11

735413

-

Brevundimonas diminuta

-

Brevundimonas diminuta D 1032

-

-

Caulobacter vibrioides NA1000

-

Caulobacter vibrioides

-

756582

A0A0H3C985

UniProt

-

UniProt

-

-

water melon, mitochondrial and glyoxysomal isoenzyme

-

-

UniProt

-

-

-

-

-

-

-

-

Comamonas testosteroni

-

Comamonas testosteroni D 1047

-

-

-

Coregonus clupeaformis

-

756858

A0A411JM49

UniProt

-

719138, 720082

-

Delftia acidovorans D 1870

basonym Alcaligenes eutrophus

-

-

-

-

Desulfurella acetivorans A63

-

Desulfurella acetivorans

-

758465

-

-

758465

-

-

-

Dictyostelium discoideum NC-4 / ATCC 24697

-

-

-

-

-

UniProt

-

-

-

UniProt

-

SwissProt

Geobacter sulfurreducens

-

657610

Q6JGI2

SwissProt

Haloarcula vallismortis

-

-

-

799, 488033

-

Haloferax volcanii DSM 3757

-

727213

D4GS06

SwissProt

sea urchin

-

strains X and Hanham

-

isopod, crustacea

-

isopod, crustacea

-

-

SwissProt

Leishmania donovani AG 83

-

-

-

-

-

-

Listeria monocytogenes

-

704320

-

bacteroids formed in symbiosis with Cicer arietinum plants

Mesorhizobium ciceri CC 1192

-

bacteroids formed in symbiosis with Cicer arietinum plants

-

Metallosphaera sedula

-

755975, 758181

Metallosphaera sedula DSM 5348

UniProt

-

755975, 758181

UniProt

Methanosarcina barkeri

-

Methylophilus methylotrophus

-

-

488044

-

-

-

Mycobacterium tuberculosis

-

UniProt

-

-

-

737214

P9WPD5

UniProt

Natronobacterium gregoryi

-

-

Natronococcus occultus

-

-

Natronomonas pharaonis

-

-

-

-

Nitrosomonas sp. TK794

-

-

-

Q9R6Y4

UniProt

-

-

cultivar Ultra

-

-

-

Priestia megaterium D101

-

-

Pseudomonas aeruginosa 8602

mutant PAC514

-

Pseudomonas chlororaphis

-

Pseudomonas chlororaphis D 302

-

-

Pseudomonas fluorescens D 1086

-

Pseudomonas fluorescens

-

662550, 703601, 777869

-

Pseudomonas fluorescens 2P24

-

777869

-

-

Pseudomonas putida NCIB 8296

-

Pseudomonas putida

-

662550, 776311

-

-

-

marine, 2 enzyme forms with different molecular weight, kinetic and regulatory properties, dissociated CS I /native CS II

-

-

Roseateles saccharophilus

-

Pyrobaculum aerophilum

-

UniProt

-

-

Roseateles saccharophilus D 1021

-

-

Saccharolobus solfataricus DSM 1617

-

757924

Saccharolobus solfataricus P1

UniProt

-

724900

-

Saccharolobus solfataricus P2

-

658613, 724900, 724945

UniProt

Saccharomyces cerevisiae MMY011

-

684713

-

Secale cereale

aluminium-tolerant cultivar Ailes and aluminium-sensitive inbred rye line Riodeva

777498

-

Sinorhizobium meliloti

-

676102

-

Solanum lycopersicum

Moneymaker

689614

-

Solanum lycopersicum Moneymaker

Moneymaker

689614

-

Solanum tuberosum

Staphylococcus aureus Newman

-

736830

-

Stenotrophomonas maltophilia

-

Stenotrophomonas maltophilia D 144

-

-

-

-

Streptomyces diastaticus SM33

UniProt

-

UniProt

Stutzerimonas stutzeri

-

-

Stutzerimonas stutzeri D 1022

-

-

-

799, 488043, 488049

-

Sulfolobus acidocaldarius 98-3 / DSM 639

-

488043, 488049

-

Sulfurisphaera tokodaii

Sulfurisphaera tokodaii DSM 16993

Sus scrofa

-

UniProt

-

-

Tetrahymena pyriformis

Tetrahymena pyriformis W

-

488063

-

Tetrahymena thermophila

Tetrahymena thermophila CU428.1

Thermus thermophilus DSM 579

Thermoplasma acidophilum 122-1B3 / ATCC 27658

-

-

-

UniProt

-

UniProt

-

-

culture condition:acetate-grown cell

-

-

-

highest expression

-

-

culture condition:glucose-grown cell

-

egg

-

-

-

-

-

-

Coregonus clupeaformis

-

-

-

-

-

-

-

-

-

-

-

-

-

-

cytosol

-

-

mitochondrial inner membrane

cytoplasmic membrane

-

isoforms CSY1 and CSY2

42579

779028

additional information

Highest Expressing Human Cell Lines

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Gene Links

malfunction

metabolism

6 entries

physiological function

Aspergillus fumigatus

Aspergillus fumigatus A1163

Desulfurella acetivorans A63

Metallosphaera sedula DSM 5348

Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis variant bovis AF2122/97

Pseudomonas aeruginosa PAO1

Thermoplasma acidophilum DSM 1728

Thermosulfidibacter takaii

Thermosulfidibacter takaii ABI70S6

100000

8 entries

105000

106000

Natronomonas pharaonis

-

gel filtration

110000

-

gel filtration

111000

-

gel filtration

112000

150000

171000

multi-angle light scattering

227000

-

gel filtration

488050

228000

-

gel filtration

240000

242000

-

sedimentation equilibrium centrifugation

249000 - 275000

-

equilibrium sedimentation

488028

250000

-

sedimentation equilibrium centrifugation

260000

-

gel filtration

269000

-

gel filtration

28000

6 * 28000, SDS-PAGE

280000

287000

-

gel filtration

300000

-

CS II, gel filtration

330000

-

gel filtration

488028

33200

-

x * 33200, calculated from amino acid sequence

36500

-

2 * 36500, CS II, SDS-PAGE

37500

-

isoenzyme CSI 6 * 37500, SDS-PAGE

40000

-

2 * 40000, SDS-PAGE

488048

40300

41000

-

2 * 41000, SDS-PAGE

42000

42600

-

2 * 42600, SDS-PAGE

42679

x * 42679, calculated from sequence

724945

43000

-

2 * 43000, SDS-PAGE

43290

2 * 43290, calculated from amino acid sequence

719429

44000

-

6 * 44000, SDS-PAGE, 6 * 49000, guanidine-HCl gel filtration

44700

-

6 * 44700, SDS-PAGE

45000

46000

-

6 * 46000, SDS-PAGE, amino-terminal amino acid sequence

47000

-

6 * 47000, SDS-PAGE, or homopentamer

488050

47885

6 * 47885, calculated from amino acid sequence

735698

48000

48700

-

2 * 48700, SDS-PAGE

49000

494770

-

gel filtration

49868

2 * 49868, MALDI-TOF mass spectrometry

50000

-

2 * 50000, CS II, SDS-PAGE

51100

Clostridium kluyveri

-

x * 51100, calculated from sequence

674333

51700

Citrus junos

Q6T8H1

x * 51700, calculated

52000

2 * 52000, SDS-PAGE

52200

x * 52200, deduced from amino acid sequence

720215

53000

58500

-

4 * 58500, sedimentation equilibrium in guanidine-HCl and dithiothreitol

60000 - 70000

-

gel filtration

60000 - 95000

-

gel filtration, sedimentation equilibrium centrifugation

66000

-

4 * 66000, SDS-PAGE

68000

-

gel filtration

69000

-

4 * 69000, sedimentation equilibrium in guanidine-HCl and dithiothreitol

73000

-

gel filtration

78000

Cicer arietinum

-

gel filtration

80000

80300

81000

-

gel filtration

83000

-

gel filtration

799, 488049

84000

85000

-

gel filtration

799, 488049

88533

Q9R6Y4

MALDI-TOF

89700

-

gel filtration

98000

Thermus aquaticus

-

gel filtration

488036

99990

gel filtration

additional information

?

dimer

dimer or oligomer

the CS4 isoform is not only present as dimer but also as high-molecular-weight oligomer under native conditions

hexamer

homodimer

homohexamer

octamer

-

8 * 55001, calculated from sequence, 8 * 70000, SDS-PAGE of recombinant protein

tetramer

additional information

13 entries

succinylation

additional information

Homo sapiens

-

SIRT3 deacetylates and activates citrate synhase activity

755951

in complex with oxaloacetate and inhibitor carboxymethyldethia-coenzyme A. The surface of citrate synthase is decoreated abundantly with basic side chains and this constellation is stable in varied pH environments

Acetobacter aceti

P20901

672091

sitting drop vapor diffusion method, using 85 mM MES pH 6.5, 10% (w/v) PEG 20000

776336

structure of apo-CSY4, at 2.69 A resolution. Dimerization of AtCSY4 is mainly mediated by alpha-helices H6, H7, H8, H12 and H13 from one molecule upon interaction with the corresponding alpha-helices from another molecule. The N-terminal (H1, H2, S1 and S2) and C-terminal (H20 and S6) regions are also important in dimerization

755738

structural modeling and functional annotation

Aspergillus niger

T1SGS1

757903

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

488085

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

-

659254

crystal structure at 1.7 A resolution. Like other Type-I CS, citrate synthase functions as a dimer and each monomer consists of a large domain and a small domain. The oxaloacetate binding site locates at the cleft between the two domains, and the active site is more closed upon binding of the oxaloacetate substrate than binding of the citrate product

Metallosphaera sedula

758181

structure of the complex with acetyl-CoA, to 1.72 A resolution. Residues His218, His258, and Asp313 are located at the active site. The pantothenic acid part of acetyl-CoA is stabilized by the main chain of Gly255 and side chain of Asp313 by direct and water mediated hydrogen binding networks. The acetyl group in the acetyl-CoA tail is stabilized by two carbonyl group at the main chain of Pro216 and Gly219 through water-mediated hydrogen bonds

Metallosphaera sedula

Mycobacterium tuberculosis

755975

hanging drop vapor diffusion method, using 0.2 M magnesium chloride hexahydrate, 0.1M Tris pH 8.5, 3.4 M 1,6-hexandiol

P9WPD5

737214

crystal structure analysis of chimeric mutants with exchange of large and small subunits between Thermoplasma acidophilum and Pyrococcus furiosus

-

hanging drop vapour diffusion method, room temperature, 2 mg/ml protein concentration, precipitation by 0.1 M sodium citrate and 0.1 M ammonium phosphate, 20 mM Tris-HCl, pH 8.0, 25 mM KCl, x-ray structure analysis, structural basis of high thermostability

488045

comparison of rigidity in citrate synthases from thermophiles to investigate the relationship between rigidity and thermostability. The increase in rigidity does not detract from the functional flexibility of the active site in all systems once their respective temperature range has been reached. In hyperthermophiles, salt bridges have stabilising roles in the active site, occuring in close proximity to key residues involved in catalysis and binding of the protein

757924

hanging-drop vapour diffusion method, 2.7 A resolution

658613

wild type enzyme bound to calcium by the hanging drop vapor diffusion method, using 0.2 M calcium acetate, 0.1 M imidazole (pH 9.0), and 4-6% (w/v) PEG 8000, and an active site variant E151Q in complex with oxaloacetate by the sitting drop vapor diffusion method, using 5% (v/v) tascimate (pH 7.0), 0.1 M HEPES (pH 7.0), 10% (w/v) PEG monomethyl ether 5000 and about 40 mM guanidine hydrochloride

structure at 2.0 A resolution. Residues His184, His259, Arg268, Arg339, and Arg359', which are important residues for substrate binding, are exposed to the inner surface of the open cleft

Sulfurisphaera tokodaii

Q974S5, Q96ZM7

756025

structure at 2.7 A resolution. Putative substrate-binding residues His180, His215, His254, Arg263, Arg335, and Arg354' are structurally conserved

Sulfurisphaera tokodaii

Q974S5, Q96ZM7

756025

pH 7.8, PEG 3350, aspartame, benzamidine, cysteamine

Sus scrofa

P00889

701519

comparison of rigidity in citrate synthases from thermophiles to investigate the relationship between rigidity and thermostability. The increase in rigidity does not detract from the functional flexibility of the active site in all systems once their respective temperature range has been reached. In hyperthermophiles, salt bridges have stabilising roles in the active site, occuring in close proximity to key residues involved in catalysis and binding of the protein

757924

crystal structure analysis of chimeric mutants with exchange of large and small subunits between Thermoplasma acidophilum and Pyrococcus furiosus

-

hanging drop vapor diffusion method, using 0.1 M CHES pH 9.5, 12% (w/v) PEG 4000, 1 mM oxaloacetate

-

unliganded form and the enzyme in complex with oxaloacetate. Oxaloacetate is most likely the quencher of tryptophan fluorescence with the oxaloacetate carbonyl as the electron acceptor

-

671987

comparison of rigidity in citrate synthases from thermophiles to investgate the relationship between rigidity and thermostability. The increase in rigidity does not detract from the functional flexibility of the active site in all systems once their respective temperature range has been reached. In hyperthermophiles, salt bridges have stabilising roles in the active site, occuring in close proximity to key residues involved in catalysis and binding of the protein

A10E

-

site-directed mutagenesis, reduced kcat, increased Km for acetyl-CoA

A361R

-

site-directed mutagenesis, slightly reduced kcat , reduced Km for acetyl-CoA and increased Km for oxaloacetate, enhanced activity with propionyl-CoA

A361R/A10E

-

site-directed mutagenesis, reduced kcat, reduced Km for acetyl-CoA

K313L

-

site-directed mutagenesis

K313L/A361R

-

site-directed mutagenesis

K313L/A361R/A10E

-

site-directed mutagenesis

A10E

-

site-directed mutagenesis, reduced kcat, increased Km for acetyl-CoA

-

A361R

-

site-directed mutagenesis, slightly reduced kcat , reduced Km for acetyl-CoA and increased Km for oxaloacetate, enhanced activity with propionyl-CoA

-

A361R/A10E

-

site-directed mutagenesis, reduced kcat, reduced Km for acetyl-CoA

-

K313L

-

site-directed mutagenesis

-

K313L/A361R

-

site-directed mutagenesis

-

A267T

D362A

-

acetyl-CoA binding site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate

D362E

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

E286D

the mutant shows reduced activity compared to the wild type enzyme

F383A

-

acetyl-CoA binding site mutant, reduced turnover

F383I

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

F383L

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

F383M

F383V

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

G181E

the mutant shows reduced activity compared to the wild type enzyme and is not inhibited by NADH

735698

H229Q

-

active site mutant, reduced turnover, increased Ki for 2-oxoglutarate

H264A

-

acetyl-CoA binding site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate

H305A

-

active site mutant, reduced turnover

K167A

-

extremely weak inhibition by NADH. Does not form hexamers in response to NADH, unlike the wild-type enzyme

659254

M372D

the mutant shows reduced activity compared to the wild type enzyme

M372K

the mutant shows reduced activity compared to the wild type enzyme

M372S

the mutant shows reduced activity compared to the wild type enzyme

R109L

-

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

659254

R163L

-

extremely weak inhibition by NADH. Does not form hexamers in response to NADH, unlike the wild-type enzyme

R306L

inactive

R314L

-

active site mutant, reduced turnover

R387L

-

active site mutant, reduced turnover

R407L

-

active site mutant, reduced turnover, increased Ki for oxaloacetate and 2-oxoglutarate

S297K

the mutant shows reduced activity compared to the wild type enzyme

S297M

the mutant shows reduced activity compared to the wild type enzyme

S297M/M372S

the mutant shows reduced activity compared to the wild type enzyme

S297T

the mutant shows reduced activity compared to the wild type enzyme

S376C

the mutant shows reduced activity compared to the wild type enzyme

S376H

the mutant shows reduced activity compared to the wild type enzyme

S376T

the mutant shows reduced activity compared to the wild type enzyme

T204R

the mutant shows reduced activity compared to the wild type enzyme and is not inhibited by NADH

735698

V361A

the mutant shows reduced activity compared to the wild type enzyme

V361A/M372S

the mutant shows reduced activity compared to the wild type enzyme

V361A/W260F

the mutant shows reduced activity compared to the wild type enzyme

V361I

the mutant shows reduced activity compared to the wild type enzyme

V361Q

the mutant shows reduced activity compared to the wild type enzyme

V361T

the mutant shows reduced activity compared to the wild type enzyme

W260F

the mutant shows reduced activity compared to the wild type enzyme

W260H

the mutant shows reduced activity compared to the wild type enzyme

W260I

the mutant shows reduced activity compared to the wild type enzyme

W260T

the mutant shows reduced activity compared to the wild type enzyme

W260V

the mutant shows reduced activity compared to the wild type enzyme

D362E

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

-

F383I

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

-

F383L

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

-

F383M

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity. The mutation not only facilitates cell growth without the presence of glutamate, but also improves the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation

-

F383V

-

the mutation reduces the enzyme's affinity for acetyl-CoA, but not eliminate its activity

-

H309G

Podospora anserina

Q9HFI1

site-directed mutagenesis, mutant allelic strain, altered developmental phenotype

488086

D113A

D113S

D177A

the mutant shows reduced activity compared to the wild type enzyme

E151Q

the mutant shows reduced activity compared to the wild type enzyme

E46Q

the mutant shows reduced activity compared to the wild type enzyme

H47A

the mutant shows reduced activity compared to the wild type enzyme

H96A

the mutant shows reduced activity compared to the wild type enzyme

R72A

the mutant shows reduced activity compared to the wild type enzyme

D12N

reference for pI-value analysis

D317G

D317 removes the acetyl-CoA methyl proton during catalysis

D317N

D317 removes the acetyl-CoA methyl proton during catalysis

G196V

-

site-directed mutagenesis, mutation interferes with dimerization, improper dimerization or dissociation of the dimer, reduced enzyme activity and conformational stability

H187Q

H222Q

H262Q

active site

R344K

-

analysis of tryptophan fluorescence

671987

S43C

-

site-directed mutagenesis, 5.7fold reduced activity, unaltered Km values for the substrates and unaltered thermostability

W245F/W115F/W17F

W348Y

additional information

22 entries

6 - 8.5

-

7

-

unstable below

100

101

Thermus aquaticus

-

recombinant enzyme, stable

488088

104

-

half-life: 1 min

40

-

stable

40 - 55

the enzyme maintains almost all of its initial activity after incubation for 20 min at 40°C or below. The half-life of the enzyme is 10 min at 50°C and is increased to 210 min in the presence of oxaloacetate. Enzyme activity is undetectable after incubation for 10 min at 55°C

44

-

melting temperature

47

half-life 4.7 min

50

55

A8YCJ7

half-life about 35 min

60

70

half-life 56.8 min

80

90

95

additional information

CS II much more stable than CS I

-

destabilization by all salts, exception is ammonium sulfate, which stabilizes at low concentration

-

dilute solutions instable, best stabilized by high concentrations of NaCl or KCl

-

488033

dithiothreitol stabilizes for about 5 days

-

freeze-drying results in 70% loss of activity

-

KCl stabilizes

-

peroxisomal enzyme protected by oxaloacetate

-

very instable without glycerol

-

very labile, 97% loss of activity after 2 d at 0°C due to aggregation

-

very labile, glycerol, 20% v/v or more stabilizes

-

-20°C, 7-10 mg/ml, 50 mM Tris buffer, pH 8.0, stable for at least 1 year

724900

-20°C, potassium phosphate buffer, pH 7.2, 15% glycerol, several weeks

-

-20°C, Tris-HCl buffer, pH 7.4, 50% glycerol, 1 month

-

-20°C, Tris-HCl buffer, pH 8.0, 1 year

-

488048

0°C, below, Tris-HCl buffer, pH 7.0, 1 mM dithiothreitol and 5 mg/ml bovine serum albumin, several months

-

4°C, 20% glycerol, 0.2mM oxaloacetate, 6 months, isoenzyme CSII stable, isoenzyme CSI 45% loss of activity

-

4°C, 25 mM Tris-HCl, pH 7.7, loss of 80% activity within 3 days, 50 mM KCl protects

-

4°C, phosphate buffer, pH 7.4, 20% loss of activity after 2 weeks

-

4°C, pure citrate synthase is completely stable for at least 6 months

-

4°C, Tris-HCl buffer, pH 7.0, 1 mM dithiothreitol, 1 day

-

4°C, Tris-HCl buffer, pH 7.6, 0.2 M KCl, 20% loss of activity after 1 month, only CS II

-

4°C, Tris-HCl buffer, pH 7.6, high protein concentration, below 20% loss of activity in 2 months

-

488033

4°C, Tris-HCl buffer, pH 8.0, 20% v/v glycerol and 0.1 M KCl, at least 6 months

-

4°C, Tris-HCl buffer, pH8.0, 50 mM glycerol, at least 1 week

-

room temperature, at 0.2 M KCl, the enzyme is stable at for at least 48 h

-

2 enzyme forms

-

amylose affinity column chromatography and HiTrap Q anion-exchange chromatography

776336

antibody studies

-

both isoenzymes

-

Cibacron Blue column chromatography and carboxymethyl Sepharose column chromatography

-

commercial preparation

Sus scrofa

P00889

701519

heat step and dye-ligand affinity chromatography

724945

HisTrap column chromatography and Source 15Q column chromatography

Matrex Red Gel A affinity column chromatography

719429

mitochondrial enzyme, antibody studies

-

488053

mitochondrial isozyme

-

488072

mitochondrial isozyme partially, glyoxysomal isozyme to homogeneity

Citrullus lanatus

-

488047

native and recombinant wild-type, the latter from E. coli and recombinant S43C mutant enzyme from E. coli

-

Ni-chelate HisTrap column chromatography

Ni-NTA bead column chromatography

-

Ni-NTA column chromatography

Mycobacterium tuberculosis

Ni-NTA column chromatography and SEC 650 gel filtration

Tetrahymena thermophila

P24118

777280

Ni-NTA column chromatography, Superdex 200 gel filtration, and Sephadex G25 gel filtration

P9WPD5

737214

Ni2+-loaded iminodiacetic acid Sepharose column chromatography and Sephadex 200 gel filtration

-

nickel-affinity column chromatography

partial

recombinant chimeric mutants from E. coli

-

recombinant chimeric protein from E. coli

recombinant enzyme from E. coli

recombinant from E. coli

-

488082

recombinant His-tagged fusion protein of citrate synthase and mitochondrial malate dehydrogenase from E. coli

-

488077

recombinant peroxisomal isozyme CS II from E. coli

-

recombinant protein

A8YCJ7

756743

recombinant protein expressed from plasmid

-

recombinant wild-type and mutant G196V from E. coli

-

recombinant wild-type, chimeric mutants, C-terminal deletion mutants, and D113 mutants from E. coli

-

recombinantz enzyme

-

recovered using dye-affinity chromatography in the presence of salt

D4GS06

727213

Talon metal (Co2+) affinity resin column chromatography

-

alfalfa is engineered by introducing the citrate synthase gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants are assayed for exclusion of aluminum from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Based on the soil assays, two transgenic events are identified that are more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance

-

689524

cloning and expression in Aspergillus nidulans, DNA sequence

Q9TEM3

488089

cloning and expression of citA in Aspergillus niger, 11fold overexpression does not increase the citrate activity level in vivo

Aspergillus niger

Q9P8D6

488074

cloning and expression of wild-type and mutants in Escherichia coli

-

cloning and sequencing

Podospora anserina

Q9HFI1

488086

cloning of His-tagged fusion protein of citrate synthase and mitochondrial malate dehydrogenase, expression in Escherichia coli

-

488077

codon-optimized expression in Escherichia coli

755738

ectopic overexpression in Arabidopsis thaliana strain WS via infection with Agrobacterium tumefaciens, DNA sequence determination

Daucus carota

O80433

expressed in Arabidopsis thaliana

-

expressed in Bacillus subtilis

Listeria monocytogenes

-

704320

expressed in Corynebacterium glutamicum strain ATCC 13032

-

737105

expressed in Escherichia coli

719429

expressed in Escherichia coli BL21 cells

expressed in Escherichia coli BL21(DE3) cells

expressed in Escherichia coli BL21(lambdaDE3) cells

expressed in Escherichia coli C41(DE3) cells

-

expressed in Escherichia coli DH5alpha cells

O00098

720215

expressed in Escherichia coli Hms174(DE3) cells

Fasciola hepatica

M4QLE7

736942

expressed in Escherichia coli Rosetta (DE3) cells

expressed in Escherichia coli using the cytoplasmic expression vectors, pET3a and pET3d. The inactive enzyme is reactivated following overnight incubation in 2 M KCl

D4GS06

727213

expressed in Nicotiana benthamiana

Citrus junos

Q6T8H1

706384

expressed in Pichia pastoris, expressed in Nicotiana tabacum

Oryza sativa

-

705651

expressed in Pseudomonas fluorescens ATCC 13525

-

705562

expression in Escherichia coli

expression in Escherichia coli BL21(DE3)

P45858

747131

expression in Escherichia coli with polyhistidine tags

P45858

747131

expression of a chimeric protein with one Acinetobacter domain in Escherichia coli, domain interactions, subunit interactions

-

expression of a chimeric protein with one Escherichia coli domain in Escherichia coli, domain interactions, subunit interactions

-

expression of chimeric mutant in Escherichia coli citrate synthase deficient strain MOB154

-

expression of labeled mitochondrial isozyme in Saccharomyces from plasmid, NMR measurements in vivo

-

488072

expression of wild-type and mutant in Escherichia coli

-

488067, 488075

expression of wild-type, chimeric mutant and site-directed mutants in Escherichia coli citrate synthase deficient strain MOB154

-

functional expression in citrate synthase deficient Escherichia coli mutant strain K214 under control of lacZ promotor, DNA and amino acid sequence analysis

Solanum tuberosum

-

488054

functional expression of peroxisomal isozyme CS II in citrate synthase deficient Escherichia coli mutant and wild-type Escherichia coli strain

-

in vitro translation of glyoxysomal isozyme, heterologous expression in Xenopus laevis oocytes

Citrullus lanatus

-

488047

mutant enzymes are expressed in Escherichia coli BL21(DE3) and C DELTApoxB cells

overexpression in Escherichia coli

724945

overexpression in Escherichia coli JM105, DNA and amino acid sequence analysis

-

overexpression of the CS gene under control of the Arabidopsis thaliana Act2 constitutive promoter or the Nicotiana tabacum RB7 root-specific promoter in alfalfa, Medicago sativa, using the Agrobacterium tumefaciens, strain LBA4404, transformation method

-

689524

the 1195-bp fragment of the mitochondrial citrate synthase enzyme is cloned in antisense orientation into the vector pBINAR between the CaMV 35S promoter and the ocs terminator. This construct is introduced into plants by an Agrobacterium

Solanum lycopersicum

-

689614

compared with the sensitive Eimeria tenella strain, the transcription of the enzyme is both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but is not significantly different in salinomycin-resistant strain

Eimeria tenella

U6KSW7

778855

CS1 is expressed differentially in nongerminated urediniospores and Pst-infected wheat leaves. The enzyme activity significantly increases upon germination compared with nongerminated urediniospores

-

enzyme expression is downregulated during iron starvation

expression is highly affected by Fe stress, indoleacetic acid and abscisic acid treatment of seedling

F4ZC76

757999

mRNA level increases in young leaf, phloem, and root, under a low iron concentration (0.004 mM) and iodoacetic acid conditions at the beginning and reaches the maximum at 12 h

-

the enzyme is inhibited during hibernation in the ground squirrel skeletal muscle, and this alteration can be mediated by decreases in succinylation

-

the enzyme transcript level rapidly increases from 0 to 24 h after the waterlogging treatment

Brassica napus

-

735413

the levels of mRNAs are reduced in response to aluminium in the roots of the tolerant and sensitive lines