Information on EC 1.2.7.11 - 2-oxoacid oxidoreductase (ferredoxin)

2-oxoacid:ferredoxin oxidoreductase

7 entries

2-oxoacid:ferredoxin oxidoreductases

3 entries

Ape1473/1472

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ordered locus name

Thermococcus kodakarensis

KOR

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OFOR

OFOR1

OFOR2

StOFOR

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StOFOR1

StOFOR2

citric acid cycle, isoleucine metabolism, threonine metabolism

a 2-oxocarboxylate + CoA + 2 oxidized ferredoxin = an acyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+

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BRENDA

KEGG

Biosynthesis of antibiotics, Biosynthesis of secondary metabolites, Butanoate metabolism, Citrate cycle (TCA cycle), Glycolysis / Gluconeogenesis, Microbial metabolism in diverse environments, Pyruvate metabolism

2-oxocarboxylate:ferredoxin 2-oxidoreductase (decarboxylating, CoA-acylating)

Contains thiamine diphosphate and [4Fe-4S] clusters [2]. This enzyme is a member of the 2-oxoacid oxidoreductases, a family of enzymes that oxidatively decarboxylate different 2-oxoacids to form their CoA derivatives, and are differentiated based on their substrate specificity. For example, see EC 1.2.7.3, 2-oxoglutarate synthase and EC 1.2.7.7, 3-methyl-2-oxobutanoate dehydrogenase (ferredoxin).

2-oxo-4-methylthiobutyrate + CoA + 2 methyl viologen

3-methylthiopropanoyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

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enzyme Ape1473/1472, 48% of the activity compared to glyoxylate

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2-oxoacid + CoA + oxidized ferredoxin

acyl-CoA + CO2 + reduced ferredoxin + H+

2-oxoadipate + CoA + 2 methyl viologen

glutaryl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

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enzyme Ape1473/1472, 49% of the activity compared to glyoxylate

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2-oxobutanoate + CoA + 2 oxidized cytochrome c

propanoyl-CoA + CO2 + 2 reduced cytochrome c + 2 H+

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2-oxobutyrate + CoA + 2 oxidized methyl viologen

propanoyl-CoA + CO2 + 2 reduced methyl viologen

2-oxobutyrate + CoA + oxidized ferredoxin

propanoyl-CoA + CO2 + reduced ferredoxin

8 entries

2-oxoglutarate + CoA + 2 oxidized cytochrome c

succinyl-CoA + CO2 + 2 reduced cytochrome c + 2 H+

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best substrate tested

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2-oxoglutarate + CoA + 2 oxidized ferredoxin

succinyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+

14 entries

2-oxoglutarate + CoA + 2 oxidized methyl viologen

succinyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

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enzyme Ape1473/1472, 94% of the activity compared to glyoxylate

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2-oxoglutarate + CoA + oxidized ferredoxin

succinyl-CoA + CO2 + reduced ferredoxin + H+

2-oxoglutarate + CoA + oxidized methyl viologen

succinyl-CoA + CO2 + reduced methyl viologen + H+

4-hydroxyphenylpyruvate + CoA + 2 methyl viologen

(4-hydroxyphenyl)acetyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

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enzyme Ape1473/1472, 16% of the activity compared to glyoxylate

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glyoxylate + CoA + 2 oxidized methyl viologen

formyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

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enzyme Ape1473/1472

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hydroxypyruvate + CoA + 2 methyl viologen

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enzyme Ape1473/1472, 55% of the activity compared to glyoxylate

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phenylpyruvate + CoA + 2 methyl viologen

phenylacetyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

pyruvate + CoA + 2 oxidized cytochrome c

acetyl-CoA + CO2 + 2 reduced cytochrome c + 2 H+

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?

pyruvate + CoA + 2 oxidized ferredoxin

acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+

18 entries

pyruvate + CoA + 2 oxidized methyl viologen

acetyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+

3 entries

pyruvate + CoA + oxidized methyl viologen

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additional information

?

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10 entries

2-oxoacid + CoA + oxidized ferredoxin

acyl-CoA + CO2 + reduced ferredoxin + H+

2-oxoglutarate + CoA + oxidized ferredoxin

succinyl-CoA + CO2 + reduced ferredoxin + H+

pyruvate + CoA + 2 oxidized ferredoxin

acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+

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additional information

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Ape1473/1472 operates in the TCA cycle of Aeropyrum pernix

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Ferredoxin

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iron-sulfur centre

the enzyme contains one [4Fe-4S]2+,1+ cluster

725876

thiamine diphosphate

7 entries

[4Fe-4S]-center

alpha/beta-subunit heterodimers contain 4Fe-4S cluster; alpha/beta-subunit heterodimers contain 4Fe-4S cluster

4Fe-4S center

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the enzyme has one [4Fe–4S]2+ cluster, ligated by 4 Cys residues, C12, C15, C46, and C197. All four Cys are required to fold a [4Fe–4S] cluster for oxidative decarboxylation of pyruvate including the formation of a stable hydroxyethyl-thiamine diphosphate radical

Iron

iron-sulfur centre

the enzyme contains one [4Fe-4S]2+,1+ cluster

725876

Mg2+

4-fluoro-7-nitrobenzofurazan

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0.001 mM inhibitor with 0.01 mM enzyme decreases the activity by about 20% and 0.003 mM inhibitor with 0.01 mM enzyme decreases the activity by around 50%. Inactivation is prevented by CoA

CoA

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substrate inhibition

KCl

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10–20% inhibition is observed with 50 mM KCl, while with higher concentrations (maximum, 0.6 M), 46–65% activation is reached, and further increased concentrations of KCl are inhibitory, enzyme Ape1473/1472

0.516

2-oxobutanoate

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pH 7.0, 55°C

0.07 - 0.516

2-oxobutyrate

0.163 - 15

2-oxoglutarate

22 entries

0.05 - 1.17

CoA

3 entries

0.07

oxidized ferredoxin

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cosubstrates: 1 mM 2-oxoxbutyrate, 0.05 mM CoA, pH and temperature not specified in the publication; cosubstrates: 1 mM pyruvate, 0.05 mM CoA, pH and temperature not specified in the publication

0.1 - 1.6

pyruvate

20 entries

additional information

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kinetic parameters of mutant enzymes

654838

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1.5

2-oxobutanoate

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pH 7.0, 55°C

1.55

2-oxobutyrate

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pH 7.0, 55°C

1.7 - 19

2-oxoglutarate

12 entries

2.2 - 51

pyruvate

12 entries

3

2-oxobutanoate

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pH 7.0, 55°C

3

2-oxobutyrate

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pH 7.0, 55°C

0.52 - 46.2

2-oxoglutarate

13 entries

7.2 - 182

pyruvate

13 entries

52.6

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pH 7.0, 55°C

6.8

assay at

7 - 8

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7

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assay at

7.5

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assay at

654838, 719399

8.5

9

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6 - 9

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pH 6.0: about 50% of maximal activity, pH 9.0: about 50% of maximal activity

7.5 - 10

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pH 7.5: about 70% of maximal activity, pH 10.0: about 70% of maximal activity, enzyme Ape1473/1472

8 - 9

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pH 8.0: 65% of maximal activity, pH 9.0: optimum, inactive below pH 7.0

50

assay at

52

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55

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assay at

70

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80

90

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110

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enzyme Ape1473/1472

25 - 52

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the enzyme is twice as active at 52°C as at 25°C

90 - 110

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70°C: 17% of maximal activity, 90°C: about 40% of maximal activity, 110°C: maximal activity, enzyme Ape1473/1472

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Desulfovibrio africanus

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740343, 740367

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Hydrogenobacter thermophilus

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740367

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674385, 740343

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Saccharolobus solfataricus DSM 1616

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Saccharolobus solfataricus P1

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beta-subunit

Q96XT4, Q96Y68

UniProt

Sulfurisphaera tokodaii DSM 16993

Thermococcus kodakarensis

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740620

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metabolism

physiological function

Sulfurisphaera tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)

34000

34410

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1 * 67405 + 1 * 34410, calculated from sequence, enzyme Ape1473/1472

37000

1 * 70000 (alpha) + 1 * 37000 (beta)

725876

42000

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2 * 86000 (alpha) + 2 * 42000 (beta), SDS-PAGE

67000

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1 * 67000 + 1 * 34000, SDS-PAGE, enzyme Ape1473/1472

67405

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1 * 67405 + 1 * 34410, calculated from sequence, enzyme Ape1473/1472

69000

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1 * 69000, alpha-subuit, + 1 * 34000, beta-subunit, SDS-PAGE and MALDI-TOF; 1 * 69000 (alpha-subunit) + 1 * 34000 (beta-subunit), SDS-PAGE, MALDI-TOF mass spectrometry

70000

71000

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1 * 71000 (subunit a) + 1 * 34000 (subunit b), SDS-PAGE

86000

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2 * 86000 (alpha) + 2 * 42000 (beta), SDS-PAGE

100000

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gel filtration, enzyme Ape1473/1472

103000

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native PAGE

105000

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gel filtration

165000

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sedimentation velocity experiments

dimer

5 entries

heterodimer

10 entries

tetramer

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2 * 86000 (alpha) + 2 * 42000 (beta), SDS-PAGE

crystals are grown at 20°C using sitting drop vapor diffusion. The structure of the recombinant enzyme StOFOR2 by the single-wavelength anomalous dispersion method is solved using a selenomethionine(SeMet)-labeled protein crystal, and the structures of the ligand-free (2.1 Å resolution) and pyruvate-complexed (2.2 Å) forms are determined. In the structure of the recombinant enzyme StOFOR2 in unreacted pyruvate complex form, the carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the alpha-subunit. The binding pockets of the 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii surrounding the methyl or propyl group of the ligands are wider than that of 2-oxoacid oxidoreductase enzymes from Desulfovbrio africanus. A possible complex structural model is constructed by placing a Zn2+-containing dicluster ferredoxin of Sulfolobus tokodaii into the large pocket of the recombinant StOFOR2 enzyme, providing insight into the electron transfer between the two redox proteins; crystals of the StOFOR1 enzyme are prepared by co-crystallization with 50 mM 2-oxobutyrate and 1 mM CoA. Crystals are grown at 25°C using sitting drop vapor diffusion. In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) is observed at the thiazole ring of thiamine diphosphate. The binding pockets of the 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii surrounding the methyl or propyl group of the ligands are wider than that of 2-oxoacid oxidoreductase enzymes from Desulfovbrio africanus; sitting drop vapor diffusion method, using 0.7 M ammonium tartrate dibasic and 0.1 M Tris-HCl (pH 8.5); sitting drop vapor diffusion method, using 15% (w/v) PEG3350, 0.1 M Tris-HCl (pH 8.5) and 0.1 M NaBr

I255L

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kcat/Km for pyruvate is 11% of wild-type value, kcat/KM for 2-oxoglutarate is 21% of wild-type value

I255M

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kcat/Km for pyruvate is 13% of wild-type value, kcat/KM for 2-oxoglutarate is 2% of wild-type value

I255S

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kcat/Km for pyruvate is 23% of wild-type value, kcat/KM for 2-oxoglutarate is 35% of wild-type value

I255V

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kcat/Km for pyruvate is 14% of wild-type value, kcat/KM for 2-oxoglutarate is 38% of wild-type value

P254G

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kcat/Km for pyruvate is 12% of wild-type value, kcat/KM for 2-oxoglutarate is 20% of wild-type value

P257A

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no enzyme activity at either 50 or 80°C

P257G

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no enzyme activity at either 50 or 80°C

P257V

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no enzyme activity at either 50 or 80°C

T256A

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kcat and Km for 2-oxoglutarate are 33% and 51%, respectively, as compared with that of the wild-type enzyme; kcat/Km for pyruvate is 21% of wild-type value, kcat/KM for 2-oxoglutarate is 15% of wild-type value

T256S

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kcat/Km for pyruvate is 15% of wild-type value, kcat/KM for 2-oxoglutarate is 92% of wild-type value

T256V

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kcat/Km for pyruvate is 17% of wild-type value, kcat/KM for 2-oxoglutarate is 68% of wild-type value

Y253A

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no enzyme activity at either 50 or 80°C

Y253F

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kcat/Km for pyruvate is 3.9% of wild-type value, kcat/KM for 2-oxoglutarate is 16% of wild-type value

Y253W

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no enzyme activity at either 50 or 80°C

T256A

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kcat and Km for 2-oxoglutarate are 33% and 51%, respectively, as compared with that of the wild-type enzyme; kcat/Km for pyruvate is 21% of wild-type value, kcat/KM for 2-oxoglutarate is 15% of wild-type value

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T256S

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kcat/Km for pyruvate is 15% of wild-type value, kcat/KM for 2-oxoglutarate is 92% of wild-type value

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T256V

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kcat/Km for pyruvate is 17% of wild-type value, kcat/KM for 2-oxoglutarate is 68% of wild-type value

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Y253F

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kcat/Km for pyruvate is 3.9% of wild-type value, kcat/KM for 2-oxoglutarate is 16% of wild-type value

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C12/15A

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loss of iron–sulfur cluster

C12A

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loss of iron–sulfur cluster. The mutant enzyme does not show formation of any radical intermediate or production of acetyl-CoA

C15A

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loss of iron–sulfur cluster. The mutant enzyme does not show formation of any radical intermediate or production of acetyl-CoA

C197A

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the enzyme retains an unidentified type of iron–sulfur cluster

C46A

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loss of iron–sulfur cluster. The mutant enzyme does not show formation of any radical intermediate or production of acetyl-CoA

D468A

inactive with 2-oxoglutarate and pyruvate as substrate; mutant enzyme StOFOR1 with mutation D468A in alpha-subunit. Vmax with pyruvate as substrate is 1.3% compared to wild-type enzyme. No activity is detected with 2-oxoglutarate

K125A

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the mutant enzyme shows a large increase in the Km-value for CoA and shows poor inactivation by 4-fluoro-7-nitrobenzofurazan, compared with K173A and wild type enzyme

K173A

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the mutant enzyme shows a large increase in the Km-value for CoA and shows poor inactivation by 4-fluoro-7-nitrobenzofurazan, compared with K173A and wild type enzyme

K49I

inactive with 2-oxoglutarate as substrate; mutant enzyme StOFOR1 with mutation K49I in alpha-subunit. Vmax with pyruvate as substrate is 28% compared to wild-type enzyme, Km with pyruvate as substrate is 2.8fold higher as compared to wild-type enzyme. No activity is detected with 2-oxoglutarate

S41A

mutant enzyme StOFOR1 with mutation S41A in alpha-subunit. Vmax with pyruvate as substrate is 29% compared to wild-type enzyme, Vmax with 2-oxoglutarate as substrate is 40% compared to wild-type enzyme, Km with pyruvate as substrate is 1.5fold higher as compared to wild-type enzyme, Km with 2-oxoglutarate as substrate is 1.5fold higher as compared to wild-type enzyme; the mutant shows reduced activity compared to the wild type enzyme

T349L

mutant enzyme StOFOR1 with mutation T349L in alpha-subunit. Vmax with pyruvate as substrate is 43% compared to wild-type enzyme, Vmax with 2-oxoglutarate as substrate is 74% compared to wild-type enzyme, Km with pyruvate as substrate is 1.6fold higher as compared to wild-type enzyme, Km with 2-oxoglutarate as substrate is 1.1fold higher as compared to wild-type enzyme; the mutant shows reduced activity compared to the wild type enzyme

D468A

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inactive with 2-oxoglutarate and pyruvate as substrate

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K49I

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inactive with 2-oxoglutarate as substrate

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S41A

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the mutant shows reduced activity compared to the wild type enzyme

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T349L

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the mutant shows reduced activity compared to the wild type enzyme

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D468A

Sulfurisphaera tokodaii DSM 16993

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mutant enzyme StOFOR1 with mutation D468A in alpha-subunit. Vmax with pyruvate as substrate is 1.3% compared to wild-type enzyme. No activity is detected with 2-oxoglutarate

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K49I

Sulfurisphaera tokodaii DSM 16993

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mutant enzyme StOFOR1 with mutation K49I in alpha-subunit. Vmax with pyruvate as substrate is 28% compared to wild-type enzyme, Km with pyruvate as substrate is 2.8fold higher as compared to wild-type enzyme. No activity is detected with 2-oxoglutarate

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S41A

Sulfurisphaera tokodaii DSM 16993

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mutant enzyme StOFOR1 with mutation S41A in alpha-subunit. Vmax with pyruvate as substrate is 29% compared to wild-type enzyme, Vmax with 2-oxoglutarate as substrate is 40% compared to wild-type enzyme, Km with pyruvate as substrate is 1.5fold higher as compared to wild-type enzyme, Km with 2-oxoglutarate as substrate is 1.5fold higher as compared to wild-type enzyme

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T349L

Sulfurisphaera tokodaii DSM 16993

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mutant enzyme StOFOR1 with mutation T349L in alpha-subunit. Vmax with pyruvate as substrate is 43% compared to wild-type enzyme, Vmax with 2-oxoglutarate as substrate is 74% compared to wild-type enzyme, Km with pyruvate as substrate is 1.6fold higher as compared to wild-type enzyme, Km with 2-oxoglutarate as substrate is 1.1fold higher as compared to wild-type enzyme

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70

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pH 7.2, 30 min, about 20% loss of activity, natural and recombinant wild-type enzyme

80

the enzymes requires high salt concentrations for stability. The half-life at 0.1 M KCl in 50 mM Tris/HC1 pH 8.0 is 3 h at 0°C

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purified enzyme is stable against oxygen

288435

-80°C, purified enzyme can be stored after dialysis against 20 mM potassium phosphate buffer, pH 6.8

288435

20°C, 4°C or -80°C, activity in cytosolic fraction is not very stable

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DEAE Sepharose column chromatography and Superdex 200 gel filtration; DEAE Sepharose column chromatography and Superdex 200 gel filtration

enzyme Ape1473/1472

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wild-type and mutant enzymes

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expressed in Escherichia coli C43 (DE3) cells; expressed in Escherichia coli C43 (DE3) cells; expression in Escherichia coli (DE3); expression in Escherichia coli (DE3)

expression in Escherichia coli, enzyme Ape1473/1472

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expression in Escherichia coli, wild-type recombinant enzyme is indistinguishable from the natural one in every criterion investigated

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