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Literature summary for 1.14.13.92 extracted from

  • Yang, G.; Cang, R.; Shen, L.; Xue, F.; Huang, H.; Zhang, Z.
    Expanding the substrate scope of phenylacetone monooxygenase from Thermobifida fusca towards cyclohexanone by protein engineering (2019), Catal. Commun., 119, 159-163 .
No PubMed abstract available

Activating Compound

Activating Compound Comment Organism Structure
additional information glucose does not improve the catalytic conversion by the enzyme Thermobifida fusca

Protein Variants

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 [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/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
phenylacetone + NADPH + H+ + O2 Thermobifida fusca
-
benzyl acetate + NADP+ + H2O
-
?

Organism

Organism UniProt Comment Textmining
Thermobifida fusca Q47PU3
-
-

Substrates and Products (Substrate)

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

Synonyms Comment Organism
PAMO
-
Thermobifida fusca

Temperature Optimum [┬░C]

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 [┬░C]

Temperature Stability Minimum [┬░C] Temperature Stability Maximum [┬░C] Comment Organism
50
-
PAMO exhibits high thermostability and activity at 50┬░C Thermobifida fusca

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Thermobifida fusca

Cofactor

Cofactor Comment Organism Structure
FAD
-
Thermobifida fusca
NADPH
-
Thermobifida fusca

General Information

General Information Comment Organism
evolution the enzyme belongs to the group II of Baeyer-Villiger monooxygenases (BMVOs) Thermobifida fusca

kcat/KM [mM/s]

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