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
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
Cdx, UniProt ID Q8VQF4, the FMN-containing redox partner to P450cin. Brownian dynamics-molecular dynamics docking method is used to produce a model of Cdx with its redox partner, enzyme P450cin, overview. Potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Arg346 plays an important role in electron transfer. Arg102 also interacts with a P450cin heme propionate. Redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. Crystal structure determination of wild-type and mutant cindoxins
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
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
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
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1.14.13.156 1,8-cineole 2-endo-monooxygenase
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