Any feedback?
Literature summary extracted from
- Directed evolution of P450cin for mediated electron transfer (2017), Protein Eng. Des. Sel., 30, 119-127 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.14.14.133 | recombinant expression of enzyme mutants in Escherichia coli strain BL21 Gold (DE3) lacIQ | Citrobacter braakii |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 1.14.14.133 | 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 |
| 1.14.14.133 | 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]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.14.133 | additional information | - |
additional information | Michaelis-Menten kinetics | Citrobacter braakii |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.14.133 | Fe2+ | in the active site heme | Citrobacter braakii |  |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.14.133 | 1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2 | Citrobacter braakii | - |
2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + H2O | - |
? | |
| 1.14.14.133 | 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
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.14.14.133 | Citrobacter braakii | Q8VQF6 | - |
- |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.14.133 | 1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2 | - |
Citrobacter braakii | 2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + H2O | - |
? | |
| 1.14.14.133 | 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 | Citrobacter braakii | ? | - |
? | |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.14.14.133 | CinA | - |
Citrobacter braakii |
| 1.14.14.133 | P450cin | - |
Citrobacter braakii |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.14.133 | cytochrome P450 | - |
Citrobacter braakii | |
| 1.14.14.133 | heme | in the active site | Citrobacter braakii | |
| 1.14.14.133 | NADPH-hemoprotein reductase | A flavoprotein containing both FMN and FAD. This enzyme catalyses the transfer of electrons from NADPH, an obligatory two-electron donor, to microsomal P-450 monooxygenases, EC 1.14.14._ | Citrobacter braakii |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.14.14.133 | 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 |
| 1.14.14.133 | 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 |
Use of this online version of BRENDA is free under the CC BY 4.0 license. See terms of use for full details.
Release 2023.1 (January 2023)
Legal
Curated at
Member of
