EC Number   |
Protein Variants |
Reference |
|---|
   1.14.14.133 | more |
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 |
746360 |
| 1.14.14.133 | N242A |
no change in characteristic CO-bound spectrum and spectrally determined KD for substrate binding. Mutation leads to modest effects on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation, but results in a reorientation of the substrate such that (R)-6'-hydroxycineole is a major product |
717775 |
| 1.14.14.133 | N242T |
significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 22% and 31%, respectively |
717156 |
| 1.14.14.133 | N242T/T243A |
significant drop in the rate of NADPH consumption. In addition to wild-type product (1R)-6beta-hydroxycineole, products (1R)-6alpha-hydroxycineole 2b and (1S)-6alpha-hydroxycineole are formed at 18% and 39%, respectively |
717156 |
| 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 |
746360 |
| 1.14.14.133 | R102A |
site-directed mutagenesis, the mutant is unable to bind the redox partner cindoxin and shows only 5% of wild-type enzyme NADPH turnover |
744316 |
| 1.14.14.133 | R346A |
site-directed mutagenesis, the mutant is unable to bind the redox partner cindoxin and shows only 10% of wild-type enzyme NADPH turnover |
744316 |
| 1.14.14.133 | T243A |
increase in the rate of NADPH consumption of 30%. Like in wild-type, single product is (1R)-6beta-hydroxycineole. T243 is not involved in controlling the protonation of the hydroperoxy species |
717156 |
