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Literature summary extracted from

  • Dudek, H.M.; Torres Pazmino, D.E.; Rodriguez, C.; de Gonzalo, G.; Gotor, V.; Fraaije, M.W.
    Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca (2010), Appl. Microbiol. Biotechnol., 88, 1135-1143.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.92 expression of His-tagged wild-type and mutant PAMOs in Escherichia coli strain TOP10 Thermobifida fusca

Protein Variants

EC Number Protein Variants Comment Organism
1.14.13.92 H220A site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220D site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220E site-directed mutagenesis, H220E mutant performs worse than wild-type PAMO with both coenzymes NADPH and NADH Thermobifida fusca
1.14.13.92 H220F site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220N site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected Thermobifida fusca
1.14.13.92 H220Q site-directed mutagenesis, the mutant shows about 3fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH is hardly affected Thermobifida fusca
1.14.13.92 H220Q/K336H site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220Q/K336N site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220T site-directed mutagenesis Thermobifida fusca
1.14.13.92 H220W site-directed mutagenesis Thermobifida fusca
1.14.13.92 K336H site-directed mutagenesis Thermobifida fusca
1.14.13.92 K336N site-directed mutagenesis Thermobifida fusca
1.14.13.92 R217A site-directed mutagenesis Thermobifida fusca
1.14.13.92 R217L site-directed mutagenesis Thermobifida fusca
1.14.13.92 T218A site-directed mutagenesis Thermobifida fusca

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.92 additional information
-
 additional information steady-state kinetics with NADPH and NADH cofactors, overview Thermobifida fusca
1.14.13.92 0.0006
-
 NADPH pH 8.0, 30°C, recombinant mutant H220N Thermobifida fusca
1.14.13.92 0.0007
-
 NADPH pH 8.0, 30°C, recombinant mutant H220A Thermobifida fusca
1.14.13.92 0.0007
-
 NADPH pH 8.0, 30°C, recombinant wild-type PAMO Thermobifida fusca
1.14.13.92 0.0011
-
 NADPH pH 8.0, 30°C, recombinant mutant K336N Thermobifida fusca
1.14.13.92 0.0014
-
 NADPH pH 8.0, 30°C, recombinant mutant T218A Thermobifida fusca
1.14.13.92 0.0017
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q Thermobifida fusca
1.14.13.92 0.0017
-
 NADPH pH 8.0, 30°C, recombinant mutant H220T Thermobifida fusca
1.14.13.92 0.0023
-
 NADPH pH 8.0, 30°C, recombinant mutant H220W Thermobifida fusca
1.14.13.92 0.0024
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336N Thermobifida fusca
1.14.13.92 0.011
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336H Thermobifida fusca
1.14.13.92 0.011
-
 NADPH pH 8.0, 30°C, recombinant mutant K336H Thermobifida fusca
1.14.13.92 0.02
-
 NADPH pH 8.0, 30°C, recombinant mutant H220F Thermobifida fusca
1.14.13.92 0.036
-
 NADPH pH 8.0, 30°C, recombinant mutant H220D Thermobifida fusca
1.14.13.92 0.17
-
 NADPH pH 8.0, 30°C, recombinant mutant H220E Thermobifida fusca
1.14.13.92 0.32
-
 NADPH pH 8.0, 30°C, recombinant mutant R217A Thermobifida fusca
1.14.13.92 0.85
-
 NADPH pH 8.0, 30°C, recombinant mutant R217L Thermobifida fusca

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.92 Thermobifida fusca
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.92 recombinant His-tagged wild-type and mutant PAMOs from Escherichia coli strain TOP10 by nickel affinity chromatography Thermobifida fusca

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.92 additional information determination of enantioselectivity of wild-type and mutant enzymes with different substrates and cofactors, overview. Residue K336 has a significant and beneficial effect on the enantioselectivity of Baeyer-Villiger oxidations and sulfoxidations Thermobifida fusca ?
-
 ?
1.14.13.92 rac-3-methyl-4-phenylbutan-2-one + NADH + H+ + O2 enantioselective reaction by PAMO Thermobifida fusca (2R)-1-phenylpropan-2-yl acetate + NAD+ + H2O
-
 ?
1.14.13.92 rac-3-methyl-4-phenylbutan-2-one + NADPH + H+ + O2 enantioselective reaction by PAMO Thermobifida fusca (2R)-1-phenylpropan-2-yl acetate + NADP+ + H2O
-
 ?
1.14.13.92 thioanisole + NADH + H+ + O2 low activity, less enantioselective reaction Thermobifida fusca thioanisole sulfoxide + NAD+ + H2O
-
 ?
1.14.13.92 thioanisole + NADPH + H+ + O2 enantioselective reaction Thermobifida fusca thioanisole sulfoxide + NADP+ + H2O mainly (R)-sulfoxide ?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.92 PAMO
-
 Thermobifida fusca

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.92 25 30 assay at Thermobifida fusca

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.92 0.8
-
 NADPH pH 8.0, 30°C, recombinant mutant H220W Thermobifida fusca
1.14.13.92 0.8
-
 NADPH pH 8.0, 30°C, recombinant mutant R217L Thermobifida fusca
1.14.13.92 1
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336N Thermobifida fusca
1.14.13.92 1.2
-
 NADPH pH 8.0, 30°C, recombinant mutant H220D Thermobifida fusca
1.14.13.92 1.2
-
 NADPH pH 8.0, 30°C, recombinant mutant H220E Thermobifida fusca
1.14.13.92 1.2
-
 NADPH pH 8.0, 30°C, recombinant mutant K336H Thermobifida fusca
1.14.13.92 1.6
-
 NADPH pH 8.0, 30°C, recombinant mutant K336N Thermobifida fusca
1.14.13.92 1.9
-
 NADPH pH 8.0, 30°C, recombinant mutant H220F Thermobifida fusca
1.14.13.92 1.9
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336H Thermobifida fusca
1.14.13.92 2.2
-
 NADPH pH 8.0, 30°C, recombinant mutant R217A Thermobifida fusca
1.14.13.92 2.7
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q Thermobifida fusca
1.14.13.92 2.9
-
 NADPH pH 8.0, 30°C, recombinant mutant H220T Thermobifida fusca
1.14.13.92 3.1
-
 NADPH pH 8.0, 30°C, recombinant wild-type PAMO Thermobifida fusca
1.14.13.92 3.3
-
 NADPH pH 8.0, 30°C, recombinant mutant H220A Thermobifida fusca
1.14.13.92 3.6
-
 NADPH pH 8.0, 30°C, recombinant mutant H220N Thermobifida fusca
1.14.13.92 3.8
-
 NADPH pH 8.0, 30°C, recombinant mutant T218A Thermobifida fusca

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.92 7.5 9 assay at Thermobifida fusca

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.92 FAD
-
 Thermobifida fusca
1.14.13.92 NADH poor activity with NADH, kinetics, binding mode, overview Thermobifida fusca
1.14.13.92 NADPH PAMO is a type I Baeyer-Villiger monooxygenase, that strongly prefers NADPH over NADH as an electron donor, the function of NADPH in catalysis cannot be easily replaced by NADH. The conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2'-phosphate moiety of NADPH, binding mode, overview. T218 and K336 do not have a strong effect on the coenzyme specificity 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
1.14.13.92 1
-
 NADPH pH 8.0, 30°C, recombinant mutant R217L Thermobifida fusca
1.14.13.92 7
-
 NADPH pH 8.0, 30°C, recombinant mutant H220E Thermobifida fusca
1.14.13.92 7
-
 NADPH pH 8.0, 30°C, recombinant mutant R217A Thermobifida fusca
1.14.13.92 33
-
 NADPH pH 8.0, 30°C, recombinant mutant H220D Thermobifida fusca
1.14.13.92 95
-
 NADPH pH 8.0, 30°C, recombinant mutant H220F Thermobifida fusca
1.14.13.92 110
-
 NADPH pH 8.0, 30°C, recombinant mutant K336H Thermobifida fusca
1.14.13.92 170
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336H Thermobifida fusca
1.14.13.92 300
-
 NADPH pH 8.0, 30°C, recombinant mutant H220W Thermobifida fusca
1.14.13.92 420
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q/K336N Thermobifida fusca
1.14.13.92 1500
-
 NADPH pH 8.0, 30°C, recombinant mutant K336N Thermobifida fusca
1.14.13.92 1600
-
 NADPH pH 8.0, 30°C, recombinant mutant H220Q Thermobifida fusca
1.14.13.92 1700
-
 NADPH pH 8.0, 30°C, recombinant mutant H220T Thermobifida fusca
1.14.13.92 2700
-
 NADPH pH 8.0, 30°C, recombinant mutant T218A Thermobifida fusca
1.14.13.92 4000
-
 NADPH pH 8.0, 30°C, recombinant wild-type PAMO Thermobifida fusca
1.14.13.92 5000
-
 NADPH pH 8.0, 30°C, recombinant mutant H220A Thermobifida fusca
1.14.13.92 6000
-
 NADPH pH 8.0, 30°C, recombinant mutant H220N Thermobifida fusca