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Literature summary extracted from

  • Loschonsky, S.; Wacker, T.; Waltzer, S.; Giovannini, P.P.; McLeish, M.J.; Andrade, S.L.; Mueller, M.
    Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: from C-C bond cleavage to C-C bond ligation (2014), Angew. Chem. Int. Ed. Engl., 53, 14402-14406.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.2.1.6 recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Azoarcus sp.
3.7.1.11 recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Azoarcus sp.

Protein Variants

EC Number Protein Variants Comment Organism
2.2.1.6 H28A site-directed mutagenesis, the mutant enzyme is much less able to catalyze the C-C bond formation as the wild-type enzyme, while the ability for C-C bond cleavage is still intact, the H28A variant shows an 8fold decrease in the formation of (R)-phenylacetylcarbinol (12%), but 1,2-diketone cleavage is nearly unaffected (78% conversion) Azoarcus sp.
2.2.1.6 H28A/N484A site-directed mutagenesis, the double mutant catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol, variant H28A/N484A shows acceptable formation of (R)-phenylacetylcarbinol (73%), but conversion toward the cleavage product is decreased by a factor of five (17% conversion), the mutant is also active with 1,2-diketone, e.g. cyclohexane-1,2-dione, in contrast to the wild-type enzyme, mutant substrate specificity amd enantioselectivity, overview Azoarcus sp.
2.2.1.6 H76A site-directed mutagenesis, almost inactive mutant Azoarcus sp.
2.2.1.6 H76A/Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.
2.2.1.6 additional information substrate specificities and enantioselectivities of wild-type and mutant enzymes, overview Azoarcus sp.
2.2.1.6 N484A site-directed mutagenesis Azoarcus sp.
2.2.1.6 Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.
3.7.1.11 H28A site-directed mutagenesis, the mutant enzyme is much less able to catalyze the C-C bond formation as the wild-type enzyme, while the ability for C-C bond cleavage is still intact, the H28A variant shows an 8fold decrease in the formation of (R)-phenylacetylcarbinol (12%), but 1,2-diketone cleavage is nearly unaffected (78% conversion) Azoarcus sp.
3.7.1.11 H28A/N484A site-directed mutagenesis, the double mutant catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol, variant H28A/N484A shows acceptable formation of (R)-phenylacetylcarbinol (73%), but conversion toward the cleavage product is decreased by a factor of five (17% conversion), the mutant is also active with 1,2-diketone in contrast to the wild-type enzyme, mutant substrate specificity amd enantioselectivity, overview Azoarcus sp.
3.7.1.11 H76A site-directed mutagenesis, inactive mutant Azoarcus sp.
3.7.1.11 H76A/Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.
3.7.1.11 additional information substrate specificities and enantioselectivities of wild-type and mutant enzymes, overview Azoarcus sp.
3.7.1.11 Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.2.1.6 Mg2+ required Azoarcus sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.2.1.6 additional information Azoarcus sp. the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, EC 3.7.1.11, and the asymmetric benzoin condensation between benzaldehyde and pyruvate ?
-
 ?
2.2.1.6 additional information Azoarcus sp. 22Lin the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, EC 3.7.1.11, and the asymmetric benzoin condensation between benzaldehyde and pyruvate ?
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O Azoarcus sp.
-
 6-oxohexanoate
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O Azoarcus sp. 22Lin
-
 6-oxohexanoate
-
 ?
3.7.1.11 additional information Azoarcus sp. the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate ?
-
 ?
3.7.1.11 additional information Azoarcus sp. 22Lin the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.2.1.6 Azoarcus sp.
-
-
-
2.2.1.6 Azoarcus sp. 22Lin
-
-
-
3.7.1.11 Azoarcus sp. P0CH62
-
-
3.7.1.11 Azoarcus sp. 22Lin P0CH62
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
3.7.1.11 cyclohexane-1,2-dione + H2O = 6-oxohexanoate the C-C bond cleavage is assumed to be initiated by the attack of the ThDP ylide on the C=O bond of the monohydrate 6 of 1,2-diketone 4 to form the ThDP adduct, a tetrahedral intermediate which breaks down to a carboxylic acid Azoarcus sp.
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.2.1.6 1-phenoxypropan-2-one + pyruvate
-
 Azoarcus sp. 3-hydroxy-3-methyl-4-phenoxybutan-2-one + CO2
-
 ?
2.2.1.6 1-phenoxypropan-2-one + pyruvate
-
 Azoarcus sp. 22Lin 3-hydroxy-3-methyl-4-phenoxybutan-2-one + CO2
-
 ?
2.2.1.6 2 butane-2,3-dione homocoupling of butane-2,3-dione by the wild-type enzyme, no activity with mutant H28A/N484A Azoarcus sp. acetylacetoin + acetoin
-
 ?
2.2.1.6 2 butane-2,3-dione homocoupling of butane-2,3-dione by the wild-type enzyme, no activity with mutant H28A/N484A Azoarcus sp. 22Lin acetylacetoin + acetoin
-
 ?
2.2.1.6 2 pyruvate in the absence of aldehydes, CDH catalyzes the decarboxylation and homocoupling of pyruvate to provide (S)-acetoin (3-hydroxybutan-2-one) with remarkably high enantioselectivity (up to 93%ee) Azoarcus sp. (S)-acetoin + 2 CO2
-
 ?
2.2.1.6 2 pyruvate in the absence of aldehydes, CDH catalyzes the decarboxylation and homocoupling of pyruvate to provide (S)-acetoin (3-hydroxybutan-2-one) with remarkably high enantioselectivity (up to 93%ee) Azoarcus sp. 22Lin (S)-acetoin + 2 CO2
-
 ?
2.2.1.6 2-acetyl-2-hydroxycyclohexanone + pyruvate
-
 Azoarcus sp. 1-(1-hydroxycyclohexyl)ethanone + CO2
-
 ?
2.2.1.6 butane-2,3-dione + benzaldehyde
-
 Azoarcus sp. ? + CO2
-
 ?
2.2.1.6 butane-2,3-dione + pyruvate
-
 Azoarcus sp. acetylacetoin + CO2
-
 ?
2.2.1.6 dihydro-2H-pyran-3(4H)-one + pyruvate
-
 Azoarcus sp. 1-(3-hydroxytetrahydro-2H-pyran-3-yl)ethanone + CO2
-
 ?
2.2.1.6 hexane-3,4-dione + pyruvate
-
 Azoarcus sp. (S)-3-ethyl-3-hydroxyhexane-2,4-dione + CO2
-
 ?
2.2.1.6 methylpyruvate + benzaldehyde
-
 Azoarcus sp. (S)-acetoin + CO2
-
 ?
2.2.1.6 additional information the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, EC 3.7.1.11, and the asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. ?
-
 ?
2.2.1.6 additional information the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, EC 3.7.1.11, and the asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. 22Lin ?
-
 ?
2.2.1.6 pyruvate + benzaldehyde asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. (R)-phenylacetylcarbinol + CO2 (R)-configuration with over 99% ee ?
2.2.1.6 pyruvate + benzaldehyde asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. 22Lin (R)-phenylacetylcarbinol + CO2 (R)-configuration with over 99% ee ?
2.2.1.6 pyruvate + cyclohexane-1,2-dione although cyclohexane-1,2-dione is a substrate of a C-C bond-cleavage reaction catalyzed by CDH, EC 3.7.1.11, wild-type CDH is unable to catalyze C-C bond formation (carboligation) using pyruvate as acyl anion donor and cyclohexane-1,2-dione as the acceptor. The formation of a tertiary alcohol is catalyzed by the enzyme double mutant CDH-H28A/N484A Azoarcus sp. ?
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O
-
 Azoarcus sp. 6-oxohexanoate
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O CDH-catalyzed C-C bond cleavage Azoarcus sp. 6-oxohexanoate
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O
-
 Azoarcus sp. 22Lin 6-oxohexanoate
-
 ?
3.7.1.11 cyclohexane-1,2-dione + H2O CDH-catalyzed C-C bond cleavage Azoarcus sp. 22Lin 6-oxohexanoate
-
 ?
3.7.1.11 additional information the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. ?
-
 ?
3.7.1.11 additional information the enzyme also catalyzes the C-C bond formation using benzaldehyde and pyruvate to form (R)-phenylacetylcarbinol, methylpyruvate or butane-2,3-dione can also serve as donor substrates. The phenylacetylcarbinol product of every active enzyme variant has (R)-configuration with over 99% ee. In the absence of aldehydes, the enzyme catalyzes the decarboxylation and homocoupling of pyruvate to provide (S)-acetoin (3-hydroxybutan-2-one) with remarkably high enantioselectivity up to 93% ee. The recombinant double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94% enantiomeric excess. The wild-type enzyme shows no activity with 1,2-diketone. Substrate specificities and enantioselectivities of wild-type and mutant enzymes, overview Azoarcus sp. ?
-
 ?
3.7.1.11 additional information the enzyme catalyzes the C-C bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. 22Lin ?
-
 ?
3.7.1.11 additional information the enzyme also catalyzes the C-C bond formation using benzaldehyde and pyruvate to form (R)-phenylacetylcarbinol, methylpyruvate or butane-2,3-dione can also serve as donor substrates. The phenylacetylcarbinol product of every active enzyme variant has (R)-configuration with over 99% ee. In the absence of aldehydes, the enzyme catalyzes the decarboxylation and homocoupling of pyruvate to provide (S)-acetoin (3-hydroxybutan-2-one) with remarkably high enantioselectivity up to 93% ee. The recombinant double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94% enantiomeric excess. The wild-type enzyme shows no activity with 1,2-diketone. Substrate specificities and enantioselectivities of wild-type and mutant enzymes, overview Azoarcus sp. 22Lin ?
-
 ?

Subunits

EC Number Subunits Comment Organism
2.2.1.6 homotetramer
-
 Azoarcus sp.

Synonyms

EC Number Synonyms Comment Organism
3.7.1.11 Cdh
-
 Azoarcus sp.
3.7.1.11 ThDP-dependent cyclohexane-1,2-dione hydrolase
-
 Azoarcus sp.
3.7.1.11 thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase
-
 Azoarcus sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.2.1.6 25 30 assay at Azoarcus sp.
3.7.1.11 25 30 assay at Azoarcus sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.2.1.6 6.5
-
 assay at Azoarcus sp.
3.7.1.11 6.5
-
 assay at Azoarcus sp.

Cofactor

EC Number Cofactor Comment Organism Structure
2.2.1.6 FAD one molecule per enzyme molecule Azoarcus sp.
2.2.1.6 thiamine diphosphate dependent on, one molecule per enzyme molecule Azoarcus sp.
3.7.1.11 thiamine diphosphate dependent on Azoarcus sp.

General Information

EC Number General Information Comment Organism
2.2.1.6 additional information wild-type and mutant H28A/N484A active site structure analysis, PDB IDs 2PGN and 4D5G Azoarcus sp.
3.7.1.11 additional information wild-type and mutant H28A/N484A active site structure analysis, PDB IDs 2PGN and 4D5G Azoarcus sp.