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Literature summary for 2.2.1.6 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
Search for more literature for 2.2.1.6

Cloned(Commentary)

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

Protein Variants

Protein Variants Comment Organism
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.
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.
H76A site-directed mutagenesis, almost inactive mutant Azoarcus sp.
H76A/Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.
additional information substrate specificities and enantioselectivities of wild-type and mutant enzymes, overview Azoarcus sp.
N484A site-directed mutagenesis Azoarcus sp.
Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.

Metals/Ions

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 ?
-
 ?
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 ?
-
 ?

Organism

Organism UniProt Comment Textmining
Azoarcus sp.
-
-
-
Azoarcus sp. 22Lin
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1-phenoxypropan-2-one + pyruvate
-
 Azoarcus sp. 3-hydroxy-3-methyl-4-phenoxybutan-2-one + CO2
-
 ?
1-phenoxypropan-2-one + pyruvate
-
 Azoarcus sp. 22Lin 3-hydroxy-3-methyl-4-phenoxybutan-2-one + CO2
-
 ?
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 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 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 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-acetyl-2-hydroxycyclohexanone + pyruvate
-
 Azoarcus sp. 1-(1-hydroxycyclohexyl)ethanone + CO2
-
 ?
butane-2,3-dione + benzaldehyde
-
 Azoarcus sp. ? + CO2
-
 ?
butane-2,3-dione + pyruvate
-
 Azoarcus sp. acetylacetoin + CO2
-
 ?
dihydro-2H-pyran-3(4H)-one + pyruvate
-
 Azoarcus sp. 1-(3-hydroxytetrahydro-2H-pyran-3-yl)ethanone + CO2
-
 ?
hexane-3,4-dione + pyruvate
-
 Azoarcus sp. (S)-3-ethyl-3-hydroxyhexane-2,4-dione + CO2
-
 ?
methylpyruvate + benzaldehyde
-
 Azoarcus sp. (S)-acetoin + CO2
-
 ?
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. ?
-
 ?
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 ?
-
 ?
pyruvate + benzaldehyde asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. (R)-phenylacetylcarbinol + CO2 (R)-configuration with over 99% ee ?
pyruvate + benzaldehyde asymmetric benzoin condensation between benzaldehyde and pyruvate Azoarcus sp. 22Lin (R)-phenylacetylcarbinol + CO2 (R)-configuration with over 99% ee ?
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. ?
-
 ?

Subunits

Subunits Comment Organism
homotetramer
-
 Azoarcus sp.

Temperature Optimum [°C]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
 assay at Azoarcus sp.

Cofactor

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

General Information

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