title: 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 KDof 2.1 ± 0.4 μM. CYP725A4 was further incorporated into nanoscale lipid bilayers (nanodiscs) and taxadiene metabolism was measured. Taxadiene metabolism followed Michaelis-Menten kinetics with an observed Vmaxof 30 ± 8 pmol/min/nmolCYP725A4and a KMof 123 ± 52 μM. 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.
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: John Edward
authors.initials: JE
authors.lastname: Rouck
authors.affiliation: Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, MA 02138, USA; Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
authors.forename: Bradley Walters
authors.initials: BW
authors.lastname: Biggs
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Amogh
authors.initials: A
authors.lastname: Kambalyal
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Arnold
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000, Belgium.
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
authors.affiliation: Manus Biosynthesis, 1030 Massachusetts Avenue, Suite 300, Cambridge, MA 02138, USA. Electronic address: pkaji@manusbio.com.
authors.forename: Parayil Kumaran
authors.initials: PK
authors.lastname: Ajikumar
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Division of Nutritional Science, Center for Biophysics and Quantitative Biology, Beckman Institute for Advanced Science and Technology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA. Electronic address: aditidas@illinois.edu.
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
mesh_terms: D000475:Alkenes / Q000737:chemistry*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004224:Diterpenes / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020946:Taxus / Q000201:enzymology / Q000235:genetics*
chemical_list: D000475:Alkenes; D004224:Diterpenes; D010940:Plant Proteins; D011994:Recombinant Proteins; C093125:taxa-4(5),11(12)diene; D003577:Cytochrome P-450 Enzyme System
keywords: E. coli; Nanodiscs; Recombinant expression; Taxadiene; Taxadiene-5α-ol