Heterologous expression and characterization of plant Taxadiene-5α-Hydroxylase (CYP725A4) in Escherichia coli.

ABSTRACT: Taxadiene-5α-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5α-ol. This oxidation is a key step in the production of the valuable cancer therapeutic and natural plant product, taxol. In this work, we report the bacterial expression and purification of six different constructs of CYP725A4. All six of these constructs are N-terminally modified and three of them are fused to cytochrome P450 reductase to form a chimera construct. The construct with the highest yield of CYP725A4 protein was then selected for substrate binding and kinetic analysis. Taxadiene binding followed type-1 substrate patterns with an observed K_D of 2.1 ± 0.4 μM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) a

SUBMITTER: Rouck JE 

PROVIDER: S-EPMC5741078 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature