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Literature summary extracted from

  • Carvalho, A.T.P.; Dourado, D.F.A.R.; Skvortsov, T.; de Abreu, M.; Ferguson, L.J.; Quinn, D.J.; Moody, T.S.; Huang, M.
    Spatial requirement for PAMO for transformation of non-native linear substrates (2018), Phys. Chem. Chem. Phys., 20, 2558-2570 .
    View publication on PubMed

Application

EC Number Application Comment Organism
1.14.13.92 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

EC Number Protein Variants Comment Organism
1.14.13.92 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
1.14.13.92 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
1.14.13.92 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
1.14.13.92 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.16 additional information
-
 additional information steady-state kinetic analysis of the mutant enzyme Thermobifida fusca
1.14.13.16 0.25
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 0.8
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 1000
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 additional information
-
 additional information steady-state kinetic analysis of wild-type and mutant enzymes Thermobifida fusca
1.14.13.92 0.06
-
 phenylacetone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.17
-
 phenylacetone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.25
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.3
-
 2-Octanone recombinant mutant R258M, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.8
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 1.4
-
 phenylacetone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 2.1
-
 2-Octanone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 3.2
-
 2-Octanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 44
-
 2-phenylcyclohexanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 1000
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 1200
-
 Cyclopentanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.16 cyclopentanone + NADPH + H+ + O2 Thermobifida fusca
-
 5-valerolactone + NADP+ + H2O
-
 ?
1.14.13.92 phenylacetone + NADPH + H+ + O2 Thermobifida fusca
-
 benzyl acetate + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.16 Thermobifida fusca
-
 phenylacetone monooxygenase enzyme mutant P253F/G254A/R258M/L443F
-
1.14.13.92 Thermobifida fusca Q47PU3
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.16 cyclopentanone + NADPH + H+ + O2 = 5-valerolactone + NADP+ + H2O the reaction mechanism of BVMO 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 Thermobifida fusca
a
1.14.13.92 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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.16 2-octanone + NADPH + H+ + O2
-
 Thermobifida fusca heptylacetate + NADP+ + H2O
-
 ?
1.14.13.16 2-phenylcyclohexanone + NADPH + H+ + O2 high activity with PAMO enzyme mutant P253F/G254A/R258M/L443F, almost no activity with wild-type PAMO (EC 1.14.13.92) Thermobifida fusca ? + NADP+ + H2O
-
 ?
1.14.13.16 cyclopentanone + NADPH + H+ + O2
-
 Thermobifida fusca 5-valerolactone + NADP+ + H2O
-
 ?
1.14.13.16 cyclopentanone + NADPH + H+ + O2 high activity with PAMO enzyme mutant P253F/G254A/R258M/L443F, almost no activity with wild-type PAMO (EC 1.14.13.92) Thermobifida fusca 5-valerolactone + NADP+ + H2O
-
 ?
1.14.13.16 additional information the engineered phenylacetone monooxygenase (EC 1.14.13.92) enzyme mutant P253F/G254A/R258M/L443F has almost lost its activity with phenylacetone, but shows higher activity with cyclopentanone instead. Substrate binding analysis, overview Thermobifida fusca ?
-
-
1.14.13.92 2-octanone + NADPH + H+ + O2
-
 Thermobifida fusca heptylacetate + NADP+ + H2O
-
 ?
1.14.13.92 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
-
 ?
1.14.13.92 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
-
 ?
1.14.13.92 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 ?
-
-
1.14.13.92 phenylacetone + NADPH + H+ + O2
-
 Thermobifida fusca benzyl acetate + NADP+ + H2O
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.16 CPMO
-
 Thermobifida fusca
1.14.13.16 More cf. EC 1.14.13.92 Thermobifida fusca
1.14.13.92 PAMO
-
 Thermobifida fusca

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.16 25
-
 assay at Thermobifida fusca
1.14.13.92 25
-
 assay at Thermobifida fusca

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.16 0.3
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 1.6
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 2.3
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.04
-
 phenylacetone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.067
-
 2-Octanone recombinant mutant R258M, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.07
-
 2-phenylcyclohexanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.091
-
 2-Octanone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.22
-
 2-Octanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.3
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.9
-
 Cyclopentanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 1.6
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 2.1
-
 phenylacetone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 2.3
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 2.4
-
 phenylacetone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.16 7.4
-
 assay at Thermobifida fusca
1.14.13.92 7.4
-
 assay at Thermobifida fusca

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.16 FAD
-
 Thermobifida fusca
1.14.13.16 NADPH
-
 Thermobifida fusca
1.14.13.92 FAD
-
 Thermobifida fusca
1.14.13.92 NADPH
-
 Thermobifida fusca

General Information

EC Number General Information Comment Organism
1.14.13.16 evolution the enzyme belongs to the group I of Baeyer-Villiger monooxygenases (BMVOs) Thermobifida fusca
1.14.13.16 additional information enzyme molecular docking and molecular dynamics, computational modeling, overview Thermobifida fusca
1.14.13.92 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
1.14.13.92 additional information enzyme molecular docking and molecular dynamics, computational modeling, overview Thermobifida fusca

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.16 0.0016
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 0.375
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.16 9.2
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.00075
-
 Cyclopentanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.0016
-
 2-phenylcyclohexanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.0016
-
 Cyclopentanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.029
-
 phenylacetone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.043
-
 2-Octanone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.069
-
 2-Octanone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.223
-
 2-Octanone recombinant mutant R258M, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 0.375
-
 2-phenylcyclohexanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 9.2
-
 2-Octanone recombinant mutant P253F/G254A/R258M/L443F, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 12.4
-
 phenylacetone recombinant mutant R258A, pH 7.4, 25°C Thermobifida fusca
1.14.13.92 40
-
 phenylacetone recombinant wild-type enzyme, pH 7.4, 25°C Thermobifida fusca