Application | Comment | Organism |
---|---|---|
synthesis | the enzyme is an ideal candidate for the synthesis of industrially relevant ester or lactone compounds. But its limited substrate scope has largely limited its industrial applications. The engineered mutant quadruple enzyme variant P253F/G254A/R258M/L443F exhibits significantly improved activity towards 2-octanone | Thermobifida fusca |
Protein Variants | Comment | Organism |
---|---|---|
additional information | rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels | Thermobifida fusca |
P253F/G254A/R258M/L443F | site-directed mutagenesis, the engineered mutant quadruple enzyme variant P253F/G254A/R258M/L443F exhibits significantly improved activity towards 2-octanone compared to wild-type. A remarkable movement of L289 is crucial for a reshaping of the active site of the quadruple variant so as to prevent the aliphatic substrate from moving away from the C4a-peroxyflavin, thus enabling it to keep a catalytically relevant pose during the oxygenation process, substrate specificity compared to wild-type | Thermobifida fusca |
R258A | site-directed mutagenesis, when the substrate 2-octanone binds to the R258A mutant, a significant change in the position of the hexyl tail of the substrate is observed, altered substrate specificity compared to wild-type | Thermobifida fusca |
R258M | site-directed mutagenesis, the R258M mutation significantly affects pose of 2-octanone, since the hexyl tail moves towards M258, substrate specificity compared to wild-type | Thermobifida fusca |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetic analysis of wild-type and mutant enzymes | Thermobifida fusca | |
0.06 | - |
phenylacetone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.17 | - |
phenylacetone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
0.25 | - |
2-Octanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
0.3 | - |
2-Octanone | recombinant mutant R258M, pH 7.4, 25°C | Thermobifida fusca | |
0.8 | - |
2-phenylcyclohexanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
1.4 | - |
phenylacetone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
2.1 | - |
2-Octanone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
3.2 | - |
2-Octanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
44 | - |
2-phenylcyclohexanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
1000 | - |
Cyclopentanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
1200 | - |
Cyclopentanone | recombinant wild-type enzyme, pH 7.4, 25°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 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
phenylacetone + NADPH + H+ + O2 = benzyl acetate + NADP+ + H2O | the reaction mechanism of BVMO, and particularly PAMO, with native substrate phenylacetone proceeds via the formation of a Criegee intermediate with anionic character, which is subsequently rearranged via the migration of alkyl group to yield the product ester, reaction mechanism, overview. Modeling of the reaction intermediate C4a-peroxyflavin | Thermobifida fusca |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-octanone + NADPH + H+ + O2 | - |
Thermobifida fusca | heptylacetate + NADP+ + H2O | - |
? | |
2-phenylcyclohexanone + NADPH + H+ + O2 | very low activity with wild-type PAMO, significantly increased activity with enzyme mutant P253F/G254A/R258M/L443F | Thermobifida fusca | ? + NADP+ + H2O | - |
? | |
cyclopentanone + NADPH + H+ + O2 | very low activity with wild-type PAMO, significantly increased activity with enzyme mutant P253F/G254A/R258M/L443F | Thermobifida fusca | 5-valerolactone + NADP+ + H2O | - |
? | |
additional information | binding of cyclopentanone and 2-phenylcyclohexanone, which are the typical substrates of CPMO in group I and CHMO in group III, respectively, is analyzed with wild-type and mutant PAMO enzymes. Substrate binding analysis, overview. Residue R337 establishes a cationn-i interaction with the substrate | 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 | Thermobifida fusca |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.04 | - |
phenylacetone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
0.067 | - |
2-Octanone | recombinant mutant R258M, pH 7.4, 25°C | Thermobifida fusca | |
0.07 | - |
2-phenylcyclohexanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.091 | - |
2-Octanone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
0.22 | - |
2-Octanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.3 | - |
2-phenylcyclohexanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
0.9 | - |
Cyclopentanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
1.6 | - |
Cyclopentanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
2.1 | - |
phenylacetone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
2.3 | - |
2-Octanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
2.4 | - |
phenylacetone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
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). The prototype of the BVMO enzyme family is the Thermobifida fusca phenylacetone monooxygenase (PAMO). Phenylacetone monooxygenase is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenases family, but it has very limited substrate scope compared with other BVMOs such as CPMO in group I or CHMO in group III | Thermobifida fusca |
additional information | enzyme molecular docking and molecular dynamics, computational modeling, overview | Thermobifida fusca |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00075 | - |
Cyclopentanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.0016 | - |
2-phenylcyclohexanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.0016 | - |
Cyclopentanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
0.029 | - |
phenylacetone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
0.043 | - |
2-Octanone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
0.069 | - |
2-Octanone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca | |
0.223 | - |
2-Octanone | recombinant mutant R258M, pH 7.4, 25°C | Thermobifida fusca | |
0.375 | - |
2-phenylcyclohexanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
9.2 | - |
2-Octanone | recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C | Thermobifida fusca | |
12.4 | - |
phenylacetone | recombinant mutant R258A, pH 7.4, 25°C | Thermobifida fusca | |
40 | - |
phenylacetone | recombinant wild-type enzyme, pH 7.4, 25°C | Thermobifida fusca |