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.

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

“…The histidine tags to construct 17-α-L-CPR and 17-α-CYP725A4 were added as published [16]. The remaining histidine tags were added using standard molecular cloning protocols.…”

“…The product was then extracted twice with 2x reaction volume of 4:1 hexane:ether and the organic extracts were combined and dried under nitrogen gas. The dried organic extract was shipped on dry ice for GC-MS analysis that was performed as previously described [16]. Products were identified by matching retention time to the retention times of previously verified structures [16].…”

“…The dried organic extract was shipped on dry ice for GC-MS analysis that was performed as previously described [16]. Products were identified by matching retention time to the retention times of previously verified structures [16]. Data were fit to the Michaelis-Menten equation.…”

“…Additionally, other groups have suggested the presence of an epoxide intermediate in the conversion of taxadiene to taxadiene-5α-ol [14, 15]. In order to help resolve the ambiguity surrounding CYP725A4-mediated taxadiene hydroxylation, we conducted a study comparing the product distribution of different CYP725A4 constructs that are purified via E. coli fermentation [16]. The use of purified protein is important because it reduces the chances for non-specific protein-protein interactions and other contaminants in the microsomes that might influence the metabolism of taxadiene by CYP725A4.…”

“…We have shown that different constructs, i.e. CYP725A4 fused to Taxus cuspidata CPR, CYP725A4 paired with mammalian CPR, and CYP725A4 paired with plant CPR, catalyzed the conversion of taxadiene to both taxadiene-5α-ol and OCT [16]. …”