BRENDA - Enzyme Database
show all sequences of 1.14.13.156

Directed evolution of P450cin for mediated electron transfer

Belsare, K.; Horn, T.; Ruff, A.; Martinez, R.; Magnusson, A.; Holtmann, D.; Schrader, J.; Schwaneberg, U.; Protein Eng. Des. Sel., 119-127 (2017)

Data extracted from this reference:

Cloned(Commentary)
Cloned (Commentary)
Organism
recombinant expression of enzyme mutants in Escherichia coli strain BL21 Gold (DE3) lacIQ
Citrobacter braakii
Engineering
Protein Variants
Commentary
Organism
additional information
construction of a functional P450 CinA-(heme center)-CinC (reductase) fusion protein separated by a linker of 10 amino acid in length, here named as P450cin-ADDCinC, to replace the multi-component system in the hydroxylation of 1,8-cineole. The P450cin-ADD-CinC variant able to hydroxylate 1,8-cineole to 2-beta-hydroxy-1,8-cineole using the alternative electron delivery systems in which zinc dust or a platinum electrode substitute the NADPH as electron source while CoIIIsep acts as electron mediator. Generation of random mutagenesis libraries and expression of mutant libraries
Citrobacter braakii
Q385H/V386S/T77N/L88R
directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems, for P450 monooxygenase P450cin: directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III) sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of sequence saturation mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generate a variant Q385H/V386S/T77N/L88R, named KB8, with a 3.8fold increase in catalytic efficiency (0.028 mM/min) compared to P450cin-ADD-CinC (0.007 mM/min). Mutant variant KB8 exhibits a 1.5fold higher product formation compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as cofactor and 4fold higher product formation rate than the P450cin-ADD-CinC mutant. Molecular docking of CoIIIsep into P450cin fusion protein
Citrobacter braakii
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Citrobacter braakii
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Fe2+
in the active site heme
Citrobacter braakii
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
1,8-cineole + NADPH + H+ + O2
Citrobacter braakii
-
2-endo-hydroxy-1,8-cineole + NADP+ + H2O
-
-
?
additional information
Citrobacter braakii
P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
?
-
-
?
Organism
Organism
UniProt
Commentary
Textmining
Citrobacter braakii
Q8VQF6
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
1,8-cineole + NADPH + H+ + O2
-
746358
Citrobacter braakii
2-endo-hydroxy-1,8-cineole + NADP+ + H2O
-
-
-
?
additional information
P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
746358
Citrobacter braakii
?
-
-
-
?
Synonyms
Synonyms
Commentary
Organism
CinA
-
Citrobacter braakii
P450cin
-
Citrobacter braakii
Cofactor
Cofactor
Commentary
Organism
Structure
cytochrome P450
-
Citrobacter braakii
heme
in the active site
Citrobacter braakii
NADPH
-
Citrobacter braakii
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant expression of enzyme mutants in Escherichia coli strain BL21 Gold (DE3) lacIQ
Citrobacter braakii
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
cytochrome P450
-
Citrobacter braakii
heme
in the active site
Citrobacter braakii
NADPH
-
Citrobacter braakii
Engineering (protein specific)
Protein Variants
Commentary
Organism
additional information
construction of a functional P450 CinA-(heme center)-CinC (reductase) fusion protein separated by a linker of 10 amino acid in length, here named as P450cin-ADDCinC, to replace the multi-component system in the hydroxylation of 1,8-cineole. The P450cin-ADD-CinC variant able to hydroxylate 1,8-cineole to 2-beta-hydroxy-1,8-cineole using the alternative electron delivery systems in which zinc dust or a platinum electrode substitute the NADPH as electron source while CoIIIsep acts as electron mediator. Generation of random mutagenesis libraries and expression of mutant libraries
Citrobacter braakii
Q385H/V386S/T77N/L88R
directed evolution to generate P450 enzymes suitable for use with alternative electron delivery systems, for P450 monooxygenase P450cin: directed evolution of a previously engineered P450 CinA-10aa-CinC fusion protein (named P450cin-ADD-CinC) to use zinc/cobalt(III) sepulchrate as electron delivery system for an increased hydroxylation activity of 1,8-cineole. Two rounds of sequence saturation mutagenesis (SeSaM) each followed by one round of multiple site-saturation mutagenesis of the P450 CinA-10aa-CinC fusion protein generate a variant Q385H/V386S/T77N/L88R, named KB8, with a 3.8fold increase in catalytic efficiency (0.028 mM/min) compared to P450cin-ADD-CinC (0.007 mM/min). Mutant variant KB8 exhibits a 1.5fold higher product formation compared to the equimolar mixture of CinA, CinC and Fpr using NADPH as cofactor and 4fold higher product formation rate than the P450cin-ADD-CinC mutant. Molecular docking of CoIIIsep into P450cin fusion protein
Citrobacter braakii
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Citrobacter braakii
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Fe2+
in the active site heme
Citrobacter braakii
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
1,8-cineole + NADPH + H+ + O2
Citrobacter braakii
-
2-endo-hydroxy-1,8-cineole + NADP+ + H2O
-
-
?
additional information
Citrobacter braakii
P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
?
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
1,8-cineole + NADPH + H+ + O2
-
746358
Citrobacter braakii
2-endo-hydroxy-1,8-cineole + NADP+ + H2O
-
-
-
?
additional information
P450cin catalyzes the stereoselective hydoxylation of 1,8-cineole to 2beta-hydroxy-1,8-cineole. The two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
746358
Citrobacter braakii
?
-
-
-
?
General Information
General Information
Commentary
Organism
evolution
P450cin from Citrobacter braakii is a three-component class I P450 system except that its flavin-containing components resemble class II P450s
Citrobacter braakii
additional information
the two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
Citrobacter braakii
General Information (protein specific)
General Information
Commentary
Organism
evolution
P450cin from Citrobacter braakii is a three-component class I P450 system except that its flavin-containing components resemble class II P450s
Citrobacter braakii
additional information
the two electrons necessary for the conversion of 1,8-cineole to 2beta-hydroxy-1,8-cineole are supplied by NADPH and transferred via a FAD-containing cindoxin reductase (CinB), and an FMN-containing cindoxin (CinC) to the heme iron in the active site of P450cin (CinA). The flow of electrons in this multicomponent P450cin system is from NADPH to Fpr via CinC to CinA
Citrobacter braakii
Other publictions for EC 1.14.13.156
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Synonyms
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
746358
Belsare
Directed evolution of P450cin ...
Citrobacter braakii
Protein Eng.
Des. Sel.
119-127
2017
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744316
Madrona
Crystal structure of cindoxin ...
Citrobacter braakii
Biochemistry
53
1435-1446
2014
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