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Literature summary for 1.14.14.1 extracted from

  • Eiben, S.; Kaysser, L.; Maurer, S.; Kuehnel, K.; Urlacher, V.B.; Schmid, R.D.
    Preparative use of isolated CYP102 monooxygenases - a critical appraisal (2006), J. Biotechnol., 124, 662-669.
    View publication on PubMed
Search for more literature for 1.14.14.1

Application

Application Comment Organism
synthesis the enzyme is intersting for regioselective production of compounds Bacillus subtilis
synthesis the enzyme is intersting for regioselective production of compounds Priestia megaterium

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli in fed-batch fermentation Bacillus subtilis
expression in Escherichia coli in fed-batch fermentation Priestia megaterium

Crystallization (Commentary)

Crystallization (Comment) Organism
crystallization of the wild-type and mutant CYP102A1 with and without bound substrates and one including theFMNbinding domain Priestia megaterium

Protein Variants

Protein Variants Comment Organism
A74E/F87V/P386S site-directed mutagenesis, the mutant shows altered regioselectivity and activity, and cofactor specificity compared to the wild-type mutant Priestia megaterium
A74G/F87V/L188Q site-directed mutagenesis, the mutant shows altered regioselectivity and activity compared to the wild-type mutant Priestia megaterium
A74G/F87V/L188Q/R966D site-directed mutagenesis, the mutant shows altered kinetics, and cofactor specificity compared to the wild-type enzyme Priestia megaterium
A74G/F87V/L188Q/R966M site-directed mutagenesis, the mutant shows altered kinetics, and cofactor specificity compared to the wild-type enzyme Priestia megaterium
A74G/F87V/L188Q/S965D site-directed mutagenesis, the mutant shows altered kinetics, and cofactor specificity compared to the wild-type enzyme Priestia megaterium
A74G/F87V/L188Q/W1046A site-directed mutagenesis, the mutant shows altered kinetics, and cofactor specificity compared to the wild-type enzyme Priestia megaterium
A74G/F87V/L188Q/W1046S site-directed mutagenesis, the mutant shows altered kinetics, and cofactor specificity compared to the wild-type enzyme Priestia megaterium
F87G site-directed mutagenesis, the mutant shows altered regioselectivity and activity compared to the wild-type mutant Priestia megaterium
F87GA site-directed mutagenesis, the mutant shows altered regioselectivity and activity compared to the wild-type mutant Priestia megaterium
additional information construction of an engineered CYP102A1 heme domain which utilizes H2O2 as electron donor instead of NADPH Priestia megaterium

Inhibitors

Inhibitors Comment Organism Structure
H2O2 at high concentrations Priestia megaterium

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.002
-
 NADPH mutants A74G/F87V/L188Q/W1046A and A74G/F87V/L188Q/W1046S Priestia megaterium
0.0025
-
 NADPH mutant A74G/F87V/L188Q Priestia megaterium
0.004
-
 NADH mutant A74G/F87V/L188Q/S965D Priestia megaterium
0.004
-
 NADH mutant enzyme A74G/F87V/L188Q/W1046A/S965D, pH and temperature not specified in the publication Priestia megaterium
0.01
-
 NADH mutant A74G/F87V/L188Q/W1046S Priestia megaterium
0.01
-
 NADH mutant enzyme A74G/F87V/L188Q/W1046S, pH and temperature not specified in the publication Priestia megaterium
0.02
-
 NADPH mutant A74G/F87V/L188Q/S965D Priestia megaterium
0.02
-
 NADH mutant A74G/F87V/L188Q/W1046A Priestia megaterium
0.02
-
 NADH mutant enzyme A74G/F87V/L188Q/W1046A, pH and temperature not specified in the publication Priestia megaterium
0.05
-
 NADPH mutant A74G/F87V/L188Q/R966M Priestia megaterium
0.13
-
 NADPH mutant A74G/F87V/L188Q/R966D Priestia megaterium
0.33
-
 NADH mutant A74G/F87V/L188Q/R966D Priestia megaterium
0.33
-
 NADH mutant enzyme A74G/F87V/L188Q/R966D, pH and temperature not specified in the publication Priestia megaterium
0.54
-
 NADH mutant A74G/F87V/L188Q/R966M Priestia megaterium
0.54
-
 NADH mutant enzyme A74G/F87V/L188Q/R966M, pH and temperature not specified in the publication Priestia megaterium
1.43
-
 NADH mutant A74G/F87V/L188Q Priestia megaterium
1.43
-
 NADH mutant enzyme A74G/F87V/L188Q, pH and temperature not specified in the publication Priestia megaterium

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ a heme-containing P450 monooxygenase Bacillus subtilis
Fe2+ a heme-containing P450 monooxygenase Priestia megaterium

Organism

Organism UniProt Comment Textmining
Bacillus subtilis
-
-
-
Priestia megaterium
-
-
-

Purification (Commentary)

Purification (Comment) Organism
several purification protocols are published, based on ion exchange chromatography, hydrophobic interaction chromatography or metal affinity chromatography, resulting in high levels of purity depending on the further application of the enzymes Bacillus subtilis
several purification protocols are published, based on ion exchange chromatography, hydrophobic interaction chromatography or metal affinity chromatography, resulting in high levels of purity depending on the further application of the enzymes Priestia megaterium

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
beta-ionone + [reduced NADPH-hemoprotein reductase] + O2
-
 Priestia megaterium 4-hydroxy-beta-ionone + [oxidized NADPH-hemoprotein reductase] + H2O
-
 ?
cytochrome c + NADH + H+ + O2
-
 Priestia megaterium ?
-
 r
cytochrome c + O2 + NADH
-
 Priestia megaterium ?
-
 r
additional information activity involves cytochrome c reduction, CYP102A1 hydroxylates and epoxidizes middle to long chain saturated, unsaturated and branched fatty acids at subterminal positions, an engineered CYP102A1 heme domain which utilizes H2O2 as electron donor Priestia megaterium ?
-
 ?
additional information activity involves cytochrome c reduction, CYP102A3 hydroxylates and epoxidizes middle to long chain saturated, unsaturated and branched fatty acids at subterminal positions Bacillus subtilis ?
-
 ?
styrene + O2 + H2O2 engineered CYP102A1 heme domain which utilizes H2O2 as electron donor instead of NADPH Priestia megaterium (R)-styrene oxide + (S)-styrene oxide + H2O
-
 ?
styrene + O2 + NAD(P)H
-
 Priestia megaterium (R)-styrene oxide + (S)-styrene oxide + H2O + NAD(P)+ enantioselective styrene oxidation with different CYP102A1 mutants, 25% S-isomer for the wild-type enzyme, 58% S-isomer for mutant A74E/F87V/P386S, 49% R-isomer for mutant F87A, 65% R-isomer for mutant A74G/F87V/L188Q, and 92% R-isomer for mutant F87G ?

Subunits

Subunits Comment Organism
More the enzyme is a natural fusion enzyme composed of an N-terminal heme monooxygenase linked to the C-terminal diflavin reductase domain Bacillus subtilis
More the enzyme is a natural fusion enzyme composed of an N-terminal heme monooxygenase linked to the C-terminal diflavin reductase domain Priestia megaterium

Synonyms

Synonyms Comment Organism
CYP102 monooxygenase
-
 Bacillus subtilis
CYP102 monooxygenase
-
 Priestia megaterium
CYP102A1
-
 Priestia megaterium
CYP102A3
-
 Bacillus subtilis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 melting temperatures of the CYP102A1 monooxygenase and the CYP102A1 reductase domain differ by about 15°C: whereas Tm for the monooxygenase domain is 63°C, it is only 48°C for the reductase domain Priestia megaterium
46
-
 10 min, half-life, wild-type CYP102A1 Priestia megaterium
61
-
 10 min, half-life, engineered CYP102A1 heme domain which utilizes H2O2 as electron donor Priestia megaterium

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.016
-
 NADH mutant A74G/F87V/L188Q Priestia megaterium
0.43
-
 NADPH mutant F87A Priestia megaterium
0.45
-
 NADPH wild-type enzyme Priestia megaterium
0.5
-
 NADPH mutant F87G Priestia megaterium
0.83
-
 NADPH mutant A74E/F87V/P386S Priestia megaterium
1.7
-
 NADPH mutant A74G/F87V/L188Q Priestia megaterium
5.67
-
 NADPH mutant A74G/F87V/L188Q/S965D Priestia megaterium
6.5
-
 NADPH mutant A74G/F87V/L188Q/W1046A Priestia megaterium
7
-
 NADH mutant A74G/F87V/L188Q/S965D Priestia megaterium
9.3
-
 NADPH mutant A74G/F87V/L188Q/R966M Priestia megaterium
9.83
-
 NADPH mutant A74G/F87V/L188Q/W1046S Priestia megaterium
32
-
 NADH mutant A74G/F87V/L188Q/R966D Priestia megaterium
37.83
-
 NADH mutant A74G/F87V/L188Q/R966M Priestia megaterium
46.83
-
 NADH mutant A74G/F87V/L188Q Priestia megaterium
89
-
 NADH mutant A74G/F87V/L188Q/W1046A Priestia megaterium
105.2
-
 NADH mutant A74G/F87V/L188Q/W1046A Priestia megaterium
132.2
-
 NADPH mutant A74G/F87V/L188Q Priestia megaterium
156
-
 NADH mutant A74G/F87V/L188Q/W1046S Priestia megaterium
183
-
 NADPH mutant A74G/F87V/L188Q/R966D Priestia megaterium
362.8
-
 NADPH mutant A74G/F87V/L188Q/R966M Priestia megaterium

Cofactor

Cofactor Comment Organism Structure
cytochrome c
-
 Bacillus subtilis
cytochrome c
-
 Priestia megaterium
FAD bound Bacillus subtilis
FAD bound, binding structure, overview Priestia megaterium
FMN bound Bacillus subtilis
FMN bound, binding structure, overview Priestia megaterium
additional information engineered CYP102A1 heme domain which utilizes H2O2 as electron donor instead of NADPH Priestia megaterium
additional information residues S965, R966 and Y974 are important in the Rossman fold-like binding motif for the cofactor binding and discrimination, overview Priestia megaterium
NAD(P)H
-
 Priestia megaterium
NADPH
-
 Bacillus subtilis