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


ABSTRACT: Taxadiene-5?-Hydroxylase (CYP725A4) is a membrane-bound plant cytochrome P450 that catalyzes the oxidation of taxadiene to taxadiene-5?-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 KD of 2.1 ± 0.4 ?M. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed Vmax of 30 ± 8 pmol/min/nmolCYP725A4 and a KM of 123 ± 52 ?M. Additionally, molecular operating environment (MOE) modeling was performed in order to gain insight into the interactions of taxadiene with CYP725A4 active site. Taken together, we demonstrate the successful expression and purification of the functional membrane-bound plant CYP, CYP725A4, in E. coli.

SUBMITTER: Rouck JE 

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

REPOSITORIES: biostudies-literature

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

Rouck John Edward JE   Biggs Bradley Walters BW   Kambalyal Amogh A   Arnold William R WR   De Mey Marjan M   Ajikumar Parayil Kumaran PK   Das Aditi A  

Protein expression and purification 20170118


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 construc  ...[more]

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