Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | glucose does not improve the catalytic conversion by the enzyme | Thermobifida fusca |
Protein Variants | Comment | Organism |
---|---|---|
I67A | site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme | Thermobifida fusca |
I67A/P440F | site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 95.1% conversion after 4 h | Thermobifida fusca |
I67C | site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme | Thermobifida fusca |
I67C/P440F | site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 92.4% conversion after 4 h | Thermobifida fusca |
I67C/P440F/A442F/L443D | site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone | Thermobifida fusca |
I67C/P440Y | site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone | Thermobifida fusca |
I67G | site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme | Thermobifida fusca |
I67G/P440F | site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 87.5% conversion after 4 h | Thermobifida fusca |
I67Y | site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme | Thermobifida fusca |
I67Y/P440F | site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 97.8% conversion after 4 h | Thermobifida fusca |
I67Y/P440Y | site-directed mutagenesis, the mutant shows high activity with cyclohexanone | Thermobifida fusca |
additional information | expanding the substrate scope of a thermostable phenylacetone monooxygenase (PAMO) to cyclohexanone by using site-directed mutagenesis. Several mutants are found to be active with cyclohexanone for which wild-type PAMO shows no activity. There is possible additive or cooperative effect existing between I67 and P440. Based on the thermostable PAMO scaffold, a chimeric PAMO-CHMO enzyme mutant is created, which shows no activity on cyclohexanone | Thermobifida fusca |
P440F | site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 50.2% conversion after 4 h | Thermobifida fusca |
P440H | site-directed mutagenesis, the mutant shows low activity with cyclohexanone | Thermobifida fusca |
P440I | site-directed mutagenesis, the mutant shows low activity with cyclohexanone | Thermobifida fusca |
P440W | site-directed mutagenesis, the mutant shows high acticity with cyclohexanone | Thermobifida fusca |
P440Y | site-directed mutagenesis, the mutant shows low activity with cyclohexanone | Thermobifida fusca |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics of wild-type and mutant enzymes | Thermobifida fusca | |
0.1037 | - |
cyclohexanone | mutant P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.2107 | - |
cyclohexanone | mutant I67Y/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.2595 | - |
cyclohexanone | mutant I67G/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.3472 | - |
cyclohexanone | mutant I67A/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.4412 | - |
cyclohexanone | mutant I67C/P440F, pH 8.0, 45°C | Thermobifida fusca |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
phenylacetone + NADPH + H+ + O2 | Thermobifida fusca | - |
benzyl acetate + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermobifida fusca | Q47PU3 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-methylcyclohexanone + NADPH + H+ + O2 | no activity with wild-type PAMO, but low to increased activity with enzyme mutants | Thermobifida fusca | ? + NADP+ + H2O | - |
? | |
4-methylcyclohexanone + NADPH + H+ + O2 | no activity with wild-type PAMO, but low to increased activity with enzyme mutants | Thermobifida fusca | ? + NADP+ + H2O | - |
? | |
cycloheptanone + NADPH + H+ + O2 | no activity with wild-type PAMO, but low to increased activity with enzyme mutants | Thermobifida fusca | ? + NADP+ + H2O | - |
? | |
cyclohexanone + NADPH + H+ + O2 | no activity with wild-type PAMO, but low to increased activity with enzyme mutants | Thermobifida fusca | epsilon-caprolactone + NADP+ + H2O | - |
? | |
cyclopentanone + NADPH + H+ + O2 | - |
Thermobifida fusca | 5-valerolactone + NADP+ + H2O | - |
? | |
additional information | substrate specificity of wild-type and mutant enzymes, overview | Thermobifida fusca | ? | - |
- |
|
phenylacetone + NADPH + H+ + O2 | - |
Thermobifida fusca | benzyl acetate + NADP+ + H2O | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PAMO | - |
Thermobifida fusca |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at, substrate phenylacetone | Thermobifida fusca |
45 | - |
assay at, substrate cyclohexanone | Thermobifida fusca |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | - |
PAMO exhibits high thermostability and activity at 50°C | Thermobifida fusca |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0456 | - |
cyclohexanone | mutant I67Y/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.0653 | - |
cyclohexanone | mutant P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.1324 | - |
cyclohexanone | mutant I67G/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.1364 | - |
cyclohexanone | mutant I67A/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.198 | - |
cyclohexanone | mutant I67C/P440F, pH 8.0, 45°C | Thermobifida fusca |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Thermobifida fusca |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
Thermobifida fusca | |
NADPH | - |
Thermobifida fusca |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the group II of Baeyer-Villiger monooxygenases (BMVOs) | Thermobifida fusca |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.216 | - |
cyclohexanone | mutant I67Y/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.393 | - |
cyclohexanone | mutant I67A/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.449 | - |
cyclohexanone | mutant I67C/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.51 | - |
cyclohexanone | mutant I67G/P440F, pH 8.0, 45°C | Thermobifida fusca | |
0.63 | - |
cyclohexanone | mutant P440F, pH 8.0, 45°C | Thermobifida fusca |