Literature summary for 1.14.14.133 extracted from

Belsare, K.; Horn, T.; Ruff, A.; Martinez, R.; Magnusson, A.; Holtmann, D.; Schrader, J.; Schwaneberg, U.
Directed evolution of P450cin for mediated electron transfer (2017), Protein Eng. Des. Sel., 30, 119-127 .

Search for more literature for 1.14.14.133

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of enzyme mutants in Escherichia coli strain BL21 Gold (DE3) lacIQ	Citrobacter braakii

Protein Variants

Protein Variants	Comment	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	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten kinetics	Citrobacter braakii

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	in the active site heme	Citrobacter braakii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2	Citrobacter braakii	-	2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + 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	Comment	Textmining
Citrobacter braakii	Q8VQF6	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
1,8-cineole + [reduced NADPH-hemoprotein reductase] + O2	-	Citrobacter braakii	2-endo-hydroxy-1,8-cineole + [oxidized NADPH-hemoprotein reductase] + 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	Citrobacter braakii	?	-	?

Synonyms

Synonyms	Comment	Organism
CinA	-	Citrobacter braakii
P450cin	-	Citrobacter braakii

Cofactor

Cofactor	Comment	Organism	Structure
cytochrome P450	-	Citrobacter braakii
heme	in the active site	Citrobacter braakii
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

General Information	Comment	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

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Release 2023.1 (January 2023)