1.14.13.92: phenylacetone monooxygenase
This is an abbreviated version!
For detailed information about phenylacetone monooxygenase, go to the full flat file.
Word Map on EC 1.14.13.92
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1.14.13.92
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baeyer-villiger
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ketone
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enantioselectivity
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bvmos
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thermobifida
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biocatalytic
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cyclohexanone
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fusca
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synthesis
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sulfoxidations
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biocatalyst
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phosphite
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cyclopentanone
- 1.14.13.92
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baeyer-villiger
- ketone
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enantioselectivity
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bvmos
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thermobifida
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biocatalytic
- cyclohexanone
- fusca
- synthesis
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sulfoxidations
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biocatalyst
- phosphite
- cyclopentanone
Reaction
Synonyms
4-hydroxyacetophenone monooxygenase, Baeyer-Villiger monooxygenase, BVMO, EtaA, HAPMO, M-PAMO, More, PAMO, phenylacetone monooxygenase, Tf PAMO
ECTree
1.14.13.92 phenylacetone monooxygenaseAdvanced search results
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results (Engineering
Engineering on EC 1.14.13.92 - phenylacetone monooxygenase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
A435Y
A442P
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 81% conversion rate
A442P/ L443I/S444Q
random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 43% conversion rate
A442P/ L443V/S444Q
random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate
A442P/L443I
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 45% conversion rate
A442P/L443L/S444Q
random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 41% conversion rate
A442P/L443T/S444Q
random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 56% conversion rate
A442P/L443V
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 90% conversion rate
A442P/L443W
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 74% conversion rate
A442P/L443W/ S444Q
random and site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 33% conversion rate
C65D
C65D/M446I/Y495I
site-directed mutagenesis, the M446I and Y495I mutations do not have significant influence on the NADPH oxidation activities. The triple mutant, which shows the greatest stability to H2O2, exhibits the highest catalytic activity (kcat) for NADPH oxidation. Thus, the oxidative stability is not markedly related to the NADPH oxidation and H2O2 generation rates
C65D/M446I/Y517I
site-directed mutagenesis, the M446I mutation does not have significant influence on the NADPH oxidation activities. The residual activity of this triple mutant variant remains unchanged during incubation with externally added H2O2. The variant completely loses the catalytic activity after 1 hour when H2O2 is generated in situ from NADPH oxidation. This fast deactivation of the C65D/M446I/Y517I variant leads to oxidation of only 10% of NADPH added. The Y517I mutation in C65D/M446I variant appears to result in blocking of the H2O2 exit and entrance path. The H2O2 generated in the active site might remain there, oxidizing amino acid residues in vicinity of the active site. The low catalytic activity of the C65D/M446I/Y517I variant for NADPH oxidation suggests that the Y517I mutation results in not only blocking of the H2O2 migration path but also modification of the active site structure
H220E
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site-directed mutagenesis, H220E mutant performs worse than wild-type PAMO with both coenzymes NADPH and NADH
H220N
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site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected
H220Q
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site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected
I67A
site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme
I67A/P440F
site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 95.1% conversion after 4 h
I67C
site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme
I67C/P440F
site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 92.4% conversion after 4 h
I67C/P440F/A442F/L443D
site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone
I67C/P440Y
site-directed mutagenesis, the mutant shows moderate activity with cyclohexanone
I67G
site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme
I67G/P440F
site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 87.5% conversion after 4 h
I67T/L338P/A435Y/A442G
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the mutant shows less than 3% of wild type activity
I67T/L338P/A435Y/A442G/L443F/S444C
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the mutant shows less than 3% of wild type activity
I67Y
site-directed mutagenesis, the mutant shows no activity with cyclohexanone like the wild-type enzyme
I67Y/P440F
site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 97.8% conversion after 4 h
I67Y/P440Y
site-directed mutagenesis, the mutant shows high activity with cyclohexanone
L153G
L443V
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate
L443V/S444M
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 53% conversion rate
L443V/S444Q
L443V/S444T
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 57% conversion rate
M446G
P253F/G254A/R258M/L443F
P440F
P440H
P440I
P440L
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440N
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440T
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440W
site-directed mutagenesis, the mutant shows high acticity with cyclohexanone
P440Y
Q93N/P94D/P440F
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at low rate
Q93W/S441A/A442G/S444C/M446G/L447P
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the mutant shows 40% of wild type activity
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
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
R337A
site-directed mutagenesis, the mutant is still able to form and stabilize the C4a-peroxyflavin intermediate, but loses the ability to convert phenylacetone or benzyle methylsulfide
R337K
site-directed mutagenesis, the mutant is still able to form and stabilize the C4a-peroxyflavin intermediate, but loses the ability to convert phenylacetone or benzyle methylsulfide
S441A/A442G/S444C/M446G/L447P
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the mutant shows 41% of wild type activity
S441D/A442E
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 73% conversion rate
S441G/A442P/L443T/S444Q
site-directed mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at about 90% conversion rate
S441G/A442T
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 48% conversion rate
S441H
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 34% conversion rate
S441H/A442P
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 78% conversion rate
V54I/C65V/I67T/Q93W/I339S/S441A/A442G/S444C/M446G/L447P
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the mutant shows 5% of wild type activity
V54I/C65V/I67T/Q93W/Q152F/I339S/S441A/A442G/S444C/M446G/L447P
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the mutant shows less than 3% of wild type activity
V54I/C65V/I67T/Q93W/Q152F/L153G/I339S/S441A/A442G/S444C/M446G/L447P
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the mutant shows less than 3% of wild type activity
W501A
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the mutant shows reduced activity compared to the wild type enzyme
additional information
C65D
site-directed mutagenesis, the engineered TfPAMO variant acts as an NADPH oxidase, it shows an extremely high rate of uncoupling compared with the wild-type enzyme. The mutant is not effective in stabilizing the C(4alpha)-peroxyflavin intermediate. For TfPAMO C65D, hydrogen peroxide and superoxide levels are highest at pH 9.0
L443V/S444Q
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 40-45% conversion rate
L443V/S444Q
random/saturation mutagenesis, the mutant is active with cyclohexanone, in contrast to the wild-type enzyme, and catalyzes its conversion to epsilon-caprolactone at 59% conversion rate
M446G
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the mutant retains wild type thermostability and produces an altered substrate binding pocket, leading to substantial changes in substrate specificity and enantioselectivity towards sulfides and ketones
M446G
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the mutant shows 49% of wild type activity and is able to convert 1-indanone to 1-isochromanone
P253F/G254A/R258M/L443F
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the mutant shows the same thermostability as the wild type enzyme while it displays an extended substrate spectrum
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
P440F
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440F
site-directed mutagenesis, the mutant shows high activity with cyclohexanone, 50.2% conversion after 4 h
P440H
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440H
site-directed mutagenesis, the mutant shows low activity with cyclohexanone
P440I
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higher subtrate variability, temperature optimum at 50C with range from 45-56C
P440I
site-directed mutagenesis, the mutant shows low activity with cyclohexanone
P440Y
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higher subtrate variability, temperature optimum at 50C with range from 45-58C
P440Y
site-directed mutagenesis, the mutant shows low activity with cyclohexanone
additional information
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construction of three mutants P1-P3 by elimination of a bulge loop region, involving residues Ser441, Ala442, and Leu443, leading to enhanced substrate enantioselectivity of Baeyer-Villiger reactions while maintaining high thermal stability, overview
additional information
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engineering of three highly stereoselective mutants of the thermally stable phenylacetone monooxygenase as practical catalysts for enantioselective Baeyer-Villiger oxidations of several ketones on a preparative scale under in vitro conditions, optimization of the method including a coupled cofactor-regeneration system, reaction mechanism, overview
additional information
directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone using iterative saturation mutagenesis, mutant screening, overview. Molecular dynamics simulations and induced fit docking of wild-type and mutant enzymes with cyclohexanone. The mutants are used in the whole cell system of Escherichia coli cells
additional information
rational engineering of enzyme PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels
additional information
a rational approach is used to improve the robustness of enzymes, in particular, Baeyer-Villiger monooxygenases (BVMOs) against H2O2. The enzyme access tunnels, which may serve as exit paths for H2O2 from the active site to the bulk, are predicted by using the CAVER and/or protein energy landscape exploration (PELE) software for mutant PAMO_C65D from Thermobifida fusca. The amino acid residues, which are susceptible to oxidation by H2O2 (e.g. methionine and tyrosine) and located in vicinity of the predicted H2O2 migration paths, are substituted with less reactive or inert amino acids (e.g. leucine and isoleucine), leading to design of H2O2-resistant enzyme variants
additional information
addition of the dimerization-docking and anchoring domain (RIDD-RIAD) system to the C-terminus of the NADPH-dependent Baeyer-Villiger monooxygenase phenylacetone monooxygenase (PAMO) and the NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH, EC 1.20.1.1) allowing self-assembly based on specific protein-protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Several RIDD/RIAD-tagged PAMO and PTDH variants are successfully overproduced in Escherichia coli and subsequently purified. Complementary tagged enzymes are mixed and analyzed for their oligomeric state, stability, and activity. Complexes are formed in the case of some specific combinations (PAMORIAD-PTDHRIDD and PAMORIAD/RIAD-PTDHRIDD). These enzyme complexes display similar catalytic activity when compared with the PTDH-PAMO fusion enzyme. The thermostability of PAMO in these complexes is retained while PTDH displays somewhat lower thermostability
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
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
