Cytochrome P450 2B1 from Rattus norvegicus
Enzyme Description
Sequence
MEPTILLLLALLVGFLLLLVRGHPKSRGNFPPGPRPLPLLGNLLQLDRGGLLNSFMQLREKYGDVFTVHLGPRPVVMLCGTDTIKEALVGQAEDFSGRGTIAVIEPIFKEYGVIFANGERWKALRRFSLATMRDFGMGKRSVEERIQEEAQCLVEELRKSQGAPLDPTFLFQCITANIICSIVFGERFDYTDRQFLRLLELFYRTFSLLSSFSSQVFEFFSGFLKYFPGAHRQISKNLQEILDYIGHIVEKHRATLDPSAPRDFIDTYLLRMEKEKSNHHTEFHHENLMISLLSLFFAGTETSSTTLRYGFLLMLKYPHVAEKVQKEIDQVIGSHRLPTLDDRSKMPYTDAVIHEIQRFSDLVPIGVPHRVTKDTMFRGYLLPKNTEVYPILSSALHDPQYFDHPDSFNPEHFLDANGALKKSEAFMPFSTGKRICLGEGIARNELFLFFTTILQNFSVSSHLAPKDIDLTPKESGIGKIPPTYQICFSAR
S. Kumar et al. (2006)
β Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide
Protein Engineering Design and Selection
Β· doi:10.1093/protein/gzl042 β
Β· Activity - Classical
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Database ID
UniProt: P00176 β
Sequence Annotation
Inferred - from protein name
Protein Source
Recombinant, host bacterium Escherichia coli TOPP3
Experimental Data (35 measurements)
35 measurements β page 2 of 2
| Mutation | Property | Assay | Solvent | Solvent Volume | Aqueous Reference | WT Reference | Measured Value | Units | Extraction method | Data type | Solution | pH | Temperature | Substrate(s) | Product(s) | Cofactor(s) | Shaking | Comments |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 5 % v/v | 100 | β | 49 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 10 % v/v | 100 | β | 33 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 15 % v/v | 100 | β | 30 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20 % v/v | 100 | β | 25 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 30 % v/v | 100 | β | 18 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 1 % v/v | 100 | β | 90 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 2.5 % v/v | 100 | β | 66 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 1 % v/v | 100 | β | 87 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 10 % v/v | 100 | β | 29 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 15 % v/v | 100 | β | 24 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 20 % v/v | 100 | β | 22 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| L209A | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 30 % v/v | 100 | β | 18 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/L295H/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 1 % v/v | 100 | β | 93 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/L295H/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 2.5 % v/v | 100 | β | 84 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
| V183L/F202L/L209A/L295H/S334P | Activity - Classical | Activity measured by fluorescence spectrophotometry (reaction product 7-hydroxy-4-trifluoromethylcoumarin fluorescence measurement, excitation at 405 nm, absorbance measurement at 510 nm) in the presence of organic solvent | Dimethyl Sulfoxide (DMSO) | 5 % v/v | 100 | β | 63 | % | Digital | Continuous | 100 mM HEPES buffer | 7.4 | Room temperature | 7-Ethoxy-4-trifluoromethylcoumarin, HβOβ (Hydrogen Peroxide) | 7-hydroxy-4-trifluoromethylcoumarin , Acetaldehyde | β | β | Classical aqueous control (in %) | H2O2 replaces the electron transfer system in the assay |
Mutations in this dataset (5)
V183L/F202L/L209A/K236I/D257N/L295H/S334P
V183L/F202L/L209A/K236I/D257N/S334P
V183L/F202L/L209A/L295H/S334P
V183L/F202L/L209A/S334P
L209A
Visualization : Activity β Classical
One bar per measurement. Colour = solvent, shade = solvent volume.