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Literature summary for 1.2.7.11 extracted from

  • Yan, Z.; Maruyama, A.; Arakawa, T.; Fushinobu, S.; Wakagi, T.
    Crystal structures of archaeal 2-oxoacid:ferredoxin oxidoreductases from Sulfolobus tokodaii (2016), Sci. Rep., 6, 33061.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.2.7.11

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli C43 (DE3) cells Sulfurisphaera tokodaii
expression in Escherichia coli (DE3) Sulfurisphaera tokodaii

Crystallization (Commentary)

Crystallization (Comment) Organism
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 Sulfurisphaera tokodaii
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 Sulfurisphaera tokodaii
sitting drop vapor diffusion method, using 0.7 M ammonium tartrate dibasic and 0.1 M Tris-HCl (pH 8.5) Sulfurisphaera tokodaii
sitting drop vapor diffusion method, using 15% (w/v) PEG3350, 0.1 M Tris-HCl (pH 8.5) and 0.1 M NaBr Sulfurisphaera tokodaii

Protein Variants

Protein Variants Comment Organism
D468A 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 Sulfurisphaera tokodaii
D468A inactive with 2-oxoglutarate and pyruvate as substrate Sulfurisphaera tokodaii
K49I 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 Sulfurisphaera tokodaii
K49I inactive with 2-oxoglutarate as substrate Sulfurisphaera tokodaii
S41A the mutant shows reduced activity compared to the wild type enzyme Sulfurisphaera tokodaii
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 Sulfurisphaera tokodaii
T349L the mutant shows reduced activity compared to the wild type enzyme Sulfurisphaera tokodaii
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 Sulfurisphaera tokodaii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.32
-
 pyruvate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, enzyme StOFOR1 Sulfurisphaera tokodaii
0.32
-
 pyruvate wild type enzyme, at pH 8.5 and 80°C Sulfurisphaera tokodaii
0.49
-
 pyruvate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, mutant enzyme StOFOR1 with mutation S41A in alpha-subunit Sulfurisphaera tokodaii
0.49
-
 pyruvate mutant enzyme S41A, at pH 8.5 and 80°C Sulfurisphaera tokodaii
0.51
-
 pyruvate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, mutant enzyme StOFOR1 with mutation T349L in alpha-subunit Sulfurisphaera tokodaii
0.51
-
 pyruvate mutant enzyme T349L, at pH 8.5 and 80°C Sulfurisphaera tokodaii
0.91
-
 pyruvate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, mutant enzyme StOFOR1 with mutation K49I in alpha-subunit Sulfurisphaera tokodaii
0.91
-
 pyruvate mutant enzyme K49I, at pH 8.5 and 80°C Sulfurisphaera tokodaii
1.6
-
 pyruvate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, enzyme StOFOR2 Sulfurisphaera tokodaii
1.6
-
 pyruvate wild type enzyme, at pH 8.5 and 80°C Sulfurisphaera tokodaii
2.1
-
 2-oxoglutarate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, enzyme StOFOR1 Sulfurisphaera tokodaii
2.1
-
 2-oxoglutarate wild type enzyme, at pH 8.5 and 80°C Sulfurisphaera tokodaii
2.3
-
 2-oxoglutarate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, mutant enzyme StOFOR1 with mutation T349L in alpha-subunit Sulfurisphaera tokodaii
2.3
-
 2-oxoglutarate mutant enzyme T349L, at pH 8.5 and 80°C Sulfurisphaera tokodaii
3.2
-
 2-oxoglutarate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, mutant enzyme StOFOR1 with mutation S41A in alpha-subunit Sulfurisphaera tokodaii
3.2
-
 2-oxoglutarate mutant enzyme S41A, at pH 8.5 and 80°C Sulfurisphaera tokodaii
15
-
 2-oxoglutarate pH 8.5, 80°C, cosubstrate: oxidized methyl viologen, enzyme StOFOR2 Sulfurisphaera tokodaii
15
-
 2-oxoglutarate wild type enzyme, at pH 8.5 and 80°C Sulfurisphaera tokodaii

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Sulfurisphaera tokodaii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
34000
-
-
 Sulfurisphaera tokodaii
70000
-
-
 Sulfurisphaera tokodaii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-oxoglutarate + CoA + oxidized ferredoxin Sulfurisphaera tokodaii
-
 succinyl-CoA + CO2 + reduced ferredoxin + H+
-
 ?
2-oxoglutarate + CoA + oxidized ferredoxin Sulfurisphaera tokodaii 7
-
 succinyl-CoA + CO2 + reduced ferredoxin + H+
-
 ?

Organism

Organism UniProt Comment Textmining
Sulfurisphaera tokodaii Q96XT2 and Q96XT4 Q96XT2: alpha-subunit (ST2435), Q96XT4: beta-subunit (ST2433); there is no evidence for the expression of the StOFOR2 (tk_24350/stk_24330 genes) in Sulfolobus tokodaii. Recombinant protein samples of STK_24350/STK_24330 are prepared and this enzyme is designated as StOFOR2
-
Sulfurisphaera tokodaii Q96XT4 beta-subunit
-
Sulfurisphaera tokodaii Q96Y66 and Q96Y68 Q96Y66: alpha-subunit (ST2300), Q96Y68: beta-subunit (ST2298)
-
Sulfurisphaera tokodaii Q96Y68 beta-subunit
-
Sulfurisphaera tokodaii 7 Q96XT4 beta-subunit
-
Sulfurisphaera tokodaii 7 Q96Y68 beta-subunit
-
Sulfurisphaera tokodaii DSM 16993 Q96XT2 and Q96XT4 Q96XT2: alpha-subunit (ST2435), Q96XT4: beta-subunit (ST2433); there is no evidence for the expression of the StOFOR2 (tk_24350/stk_24330 genes) in Sulfolobus tokodaii. Recombinant protein samples of STK_24350/STK_24330 are prepared and this enzyme is designated as StOFOR2
-
Sulfurisphaera tokodaii DSM 16993 Q96Y66 and Q96Y68 Q96Y66: alpha-subunit (ST2300), Q96Y68: beta-subunit (ST2298)
-

Purification (Commentary)

Purification (Comment) Organism
-
 Sulfurisphaera tokodaii
DEAE Sepharose column chromatography and Superdex 200 gel filtration Sulfurisphaera tokodaii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-oxobutyrate + CoA + 2 oxidized methyl viologen
-
 Sulfurisphaera tokodaii propanoyl-CoA + CO2 + 2 reduced methyl viologen
-
 ?
2-oxobutyrate + CoA + 2 oxidized methyl viologen
-
 Sulfurisphaera tokodaii 7 propanoyl-CoA + CO2 + 2 reduced methyl viologen
-
 ?
2-oxobutyrate + CoA + 2 oxidized methyl viologen
-
 Sulfurisphaera tokodaii DSM 16993 propanoyl-CoA + CO2 + 2 reduced methyl viologen
-
 ?
2-oxoglutarate + CoA + 2 oxidized ferredoxin because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii succinyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
2-oxoglutarate + CoA + 2 oxidized ferredoxin the recombinant enzyme StOFOR2 exhibits a preference for pyruvate over 2-oxoglutarate. Because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii succinyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
2-oxoglutarate + CoA + 2 oxidized ferredoxin the recombinant enzyme StOFOR2 exhibits a preference for pyruvate over 2-oxoglutarate. Because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii DSM 16993 succinyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
2-oxoglutarate + CoA + 2 oxidized ferredoxin because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii DSM 16993 succinyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
2-oxoglutarate + CoA + oxidized ferredoxin
-
 Sulfurisphaera tokodaii succinyl-CoA + CO2 + reduced ferredoxin + H+
-
 ?
2-oxoglutarate + CoA + oxidized ferredoxin
-
 Sulfurisphaera tokodaii 7 succinyl-CoA + CO2 + reduced ferredoxin + H+
-
 ?
2-oxoglutarate + CoA + oxidized methyl viologen
-
 Sulfurisphaera tokodaii succinyl-CoA + CO2 + reduced methyl viologen + H+
-
 ?
2-oxoglutarate + CoA + oxidized methyl viologen
-
 Sulfurisphaera tokodaii 7 succinyl-CoA + CO2 + reduced methyl viologen + H+
-
 ?
pyruvate + CoA + 2 oxidized ferredoxin because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
pyruvate + CoA + 2 oxidized ferredoxin the recombinant enzyme StOFOR2 exhibits a preference for pyruvate over 2-oxoglutarate. The carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the alpha-subunit of Sulfurisphaera tokodaii acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
pyruvate + CoA + 2 oxidized ferredoxin the recombinant enzyme StOFOR2 exhibits a preference for pyruvate over 2-oxoglutarate. The carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the alpha-subunit of Sulfurisphaera tokodaii DSM 16993 acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
pyruvate + CoA + 2 oxidized ferredoxin because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii DSM 16993 acetyl-CoA + CO2 + 2 reduced ferredoxin + 2 H+
-
 ?
pyruvate + CoA + 2 oxidized methyl viologen
-
 Sulfurisphaera tokodaii acetyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+
-
 ?
pyruvate + CoA + 2 oxidized methyl viologen
-
 Sulfurisphaera tokodaii DSM 16993 acetyl-CoA + CO2 + 2 reduced methyl viologen + 2 H+
-
 ?
pyruvate + CoA + oxidized methyl viologen
-
 Sulfurisphaera tokodaii ?
-
 ?
pyruvate + CoA + oxidized methyl viologen
-
 Sulfurisphaera tokodaii 7 ?
-
 ?

Subunits

Subunits Comment Organism
heterodimer crystals consist of two protomers (1 and 2), each of which consists of one heterodimer of alpha- and beta-subunits Sulfurisphaera tokodaii
heterodimer 1 * 70000 + 1 * 34000, SDS-PAGE Sulfurisphaera tokodaii

Synonyms

Synonyms Comment Organism
2-oxoacid:ferredoxin oxidoreductases
-
 Sulfurisphaera tokodaii
OFOR1 isoform Sulfurisphaera tokodaii
OFOR2 isoform Sulfurisphaera tokodaii
StOFOR1
-
 Sulfurisphaera tokodaii
StOFOR1 StOFOR1 and StOFOR2 are paralogs, the 2-oxoacid oxidoreductase activity in Sulfolobus tokodaii cells is mainly due to StOFOR1. A paralogous gene set (stk_24350/stk_24330) of the StOFOR1 genes (stk_23000/stk_22980) is present in the Sulfolobus tokodaii genome. STK_24350 (alpha-subunit) and STK_24330 (beta-subunit) have similar domain architectures to STK_23000 and STK_22980, with high amino acid sequence identity for both subunits (59% and 71%). 2-oxoacid oxidoreductase activity in Sulfolobus tokodaii cells is mainly due to StOFOR1, and there is no evidence for the expression of the tk_24350/stk_24330 genes Sulfurisphaera tokodaii
StOFOR2
-
 Sulfurisphaera tokodaii
StOFOR2 there is no evidence for the expression of the StOFOR2 (tk_24350/stk_24330 genes) in Sulfolobus tokodaii. Recombinant protein samples of STK_24350/STK_24330 are prepared and this enzyme is designated as StOFOR2. StOFOR1 and StOFOR2 are paralog. The 2-oxoacid oxidoreductase activity in Sulfolobus tokodaii cells is mainly due to StOFOR1. A paralogous gene set (stk_24350/stk_24330) of the StOFOR1 genes (stk_23000/stk_22980) is present in the Sulfolobus tokodaii genome. STK_24350 (alpha-subunit) and STK_24330 (beta-subunit) have similar domain architectures to STK_23000 and STK_22980, with high amino acid sequence identity for both subunits (59% and 71%) Sulfurisphaera tokodaii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
 assay at Sulfurisphaera tokodaii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 assay at Sulfurisphaera tokodaii

Cofactor

Cofactor Comment Organism Structure
Ferredoxin alpha/beta-subunit heterodimers lack an intramolecular ferredoxin domain. Because 2-oxoacid oxidoreductase enzymes from Sulfolobus tokodaii lack the intramolecular Fd-like domain V, there is a large pocket surrounded by domains III and VI in each protomer, which appears to be able to bind an external ferredoxin molecule Sulfurisphaera tokodaii
thiamine diphosphate
-
 Sulfurisphaera tokodaii
thiamine diphosphate alpha/beta-subunit heterodimers contain thiamin diphosphate Sulfurisphaera tokodaii
[4Fe-4S]-center
-
 Sulfurisphaera tokodaii
[4Fe-4S]-center alpha/beta-subunit heterodimers contain 4Fe-4S cluster Sulfurisphaera tokodaii

General Information

General Information Comment Organism
metabolism can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism Sulfurisphaera tokodaii
metabolism there is no evidence for the expression of the StOFOR2 (tk_24350/stk_24330 genes) in Sulfolobus tokodaii. It may be expressed in vivo under some culture conditions Sulfurisphaera tokodaii