Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4
2016
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 togethe...
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