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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Azoarcus sp. |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Azoarcus sp. | - |
- |
- |
Azoarcus sp. 22Lin | - |
- |
- |
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 | Comment | Organism |
---|---|---|
homotetramer | - |
Azoarcus sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | 30 | assay at | Azoarcus sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.5 | - |
assay at | Azoarcus sp. |
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 | Comment | Organism |
---|---|---|
additional information | wild-type and mutant H28A/N484A active site structure analysis, PDB IDs 2PGN and 4D5G | Azoarcus sp. |