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Literature summary for 3.7.1.11 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 3.7.1.11

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 in contrast to the wild-type enzyme, mutant substrate specificity amd enantioselectivity, overview Azoarcus sp.
H76A site-directed mutagenesis, 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.
Q116A site-directed mutagenesis, inactive mutant Azoarcus sp.

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
cyclohexane-1,2-dione + H2O Azoarcus sp.
-
 6-oxohexanoate
-
 ?
cyclohexane-1,2-dione + H2O Azoarcus sp. 22Lin
-
 6-oxohexanoate
-
 ?
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 ?
-
 ?
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

Organism UniProt Comment Textmining
Azoarcus sp. P0CH62
-
-
Azoarcus sp. 22Lin P0CH62
-
-

Reaction

Reaction Comment Organism Reaction ID
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cyclohexane-1,2-dione + H2O
-
 Azoarcus sp. 6-oxohexanoate
-
 ?
cyclohexane-1,2-dione + H2O CDH-catalyzed C-C bond cleavage Azoarcus sp. 6-oxohexanoate
-
 ?
cyclohexane-1,2-dione + H2O
-
 Azoarcus sp. 22Lin 6-oxohexanoate
-
 ?
cyclohexane-1,2-dione + H2O CDH-catalyzed C-C bond cleavage Azoarcus sp. 22Lin 6-oxohexanoate
-
 ?
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. ?
-
 ?
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. ?
-
 ?
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 ?
-
 ?
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 ?
-
 ?

Synonyms

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