title: Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4.
abstract: Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzyme's inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxol's biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts.
authors.affiliation: Manus Biosynthesis , 1030 Massachusetts Avenue, Suite 300, Cambridge, Massachusetts 02138, United States.|Department of Chemical and Biological Engineering (Masters in Biotechnology Program), Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Bradley Walters
authors.initials: BW
authors.lastname: Biggs
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute for Advanced Science and Engineering, University of Illinois Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: John Edward
authors.initials: JE
authors.lastname: Rouck
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute for Advanced Science and Engineering, University of Illinois Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Amogh
authors.initials: A
authors.lastname: Kambalyal
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute for Advanced Science and Engineering, University of Illinois Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: William
authors.initials: W
authors.lastname: Arnold
authors.affiliation: Manus Biosynthesis , 1030 Massachusetts Avenue, Suite 300, Cambridge, Massachusetts 02138, United States.
authors.forename: Chin Giaw
authors.initials: CG
authors.lastname: Lim
authors.affiliation: Manus Biosynthesis , 1030 Massachusetts Avenue, Suite 300, Cambridge, Massachusetts 02138, United States.|Centre for Industrial Biotechnology and Biocatalysis, Ghent University , Coupure Links 653, B-9000, Ghent, Belgium.
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
authors.affiliation: Sequoia Sciences , 1912 Innerbelt Business Center Dr., Saint Louis, Missouri 63114, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: O'Neil-Johnson
authors.affiliation: Sequoia Sciences , 1912 Innerbelt Business Center Dr., Saint Louis, Missouri 63114, United States.
authors.forename: Courtney M
authors.initials: CM
authors.lastname: Starks
authors.affiliation: Department of Comparative Biosciences, Department of Biochemistry, Department of Bioengineering, Beckman Institute for Advanced Science and Engineering, University of Illinois Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Aditi
authors.initials: A
authors.lastname: Das
authors.affiliation: Manus Biosynthesis , 1030 Massachusetts Avenue, Suite 300, Cambridge, Massachusetts 02138, United States.
authors.forename: Parayil Kumaran
authors.initials: PK
authors.lastname: Ajikumar
mesh_terms: D000475:Alkenes / Q000737:chemistry / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000378:metabolism*; D053898:Biosynthetic Pathways; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D005285:Fermentation; D060847:Metabolic Engineering; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017239:Paclitaxel / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D020946:Taxus / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
chemical_list: D000475:Alkenes; D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; C093125:taxa-4(5),11(12)diene; D003577:Cytochrome P-450 Enzyme System; D017239:Paclitaxel