Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase

Abstract

The anticancer molecule taxol (Paclitaxel) stands as one of the most medically and economically important natural products. However, despite decades of extensive study, its biosynthesis remains poorly understood. Unpredictable behavior of the first oxygenation enzyme, taxadiene-5α-hydroxylase, which produces a range of undesired products, currently stands as a key bottleneck to improved taxol production. We herein present chemical and biological evidence of an unreported epoxidase activity of taxadiene-5α-hydroxylase that puts into question the previously proposed radical-rebound mechanism. We demonstrate that the poor selectivity of taxadiene-5α-hydroxylase arises from nonselective degradation of an epoxide intermediate produced via a selective oxidation step, rather than from promiscuous oxidation, as previously proposed. We support these conclusions by demonstrating variable enzyme behavior in differing hosts and conditions, similarity of products and product ratios generated from chemical epoxidation, and taxadiene-5α-hydroxylase, and differing enzymatic activity on alternative taxadiene isomers. Additionally, we use directed mutagenesis to describe the oxidizing species of the P450, demonstrate that further in vivo functionalization of oxidized taxadiene is unable to improve selectivity of the oxidation, and show that multiple products are produced in the Taxus cuspidata and are not simply an artifact of heterologous expression. Our results highlight an important, and previously unknown, obstacle to improved taxol production. We further offer insights to overcome the challenges posed by an epoxide-mediated reaction, which sets the basis for further engineering of taxol biosynthesis.

Two-step pathway for isoprenoid synthesis

“…Cell density was monitored by UV/Vis spectroscopy at 600 nm while glucose consumption was determined by HPLC using an Aminex HPX-87H column (300 × 7.8 mm) (Bio-Rad) on an Infinity 1260 series HPLC (Agilent) at a flow rate of 0.7 mL/min with 14 mM H 2 SO 4 , at room temperature using a refractive index detector set at 50°C. C18 flash resin was added to taxadiene bioreactors to capture taxadiene and eluted in acetonitrile for purification by semipreparative HPLC as previously described (33).…”

P450s controlling metabolic bifurcations in plant terpene specialized metabolism

“…Based on the prevalent distribution of the pattern of hydroxylation, the next predicted step requires the P450 taxadiene-5α-hydroxylase, CYP725A4, which catalyzes the regio-selective hydroxylation of the 5α position of taxadiene to yield taxa-4(20),11(12)-dien-5α-ol (T5OH/T-5α-ol) (Fig. 10 , Biggs et al 2016b ; Edgar et al 2016 ; Hefner et al 1996 ; Jennewein et al 2004 ). The subsequent conversions include a series of P450-mediated oxygenations, providing the molecular handles for acylations and further conjugation of the skeleton.…”

“…It has been observed that the heterologous expression of taxadiene synthase and CYP725A4 in tobacco ( Nicotiana sylvestris ) result in the formation of 5(12)-oxa-3(11)-cyclotaxane (OCT) instead of T5OH (Rontein et al 2008 ). Further studies described that expression of recombinant taxadiene synthase and CYP725A4 in an E. coli host leads to equal formation of T5OH and OCT and indicated that the lack of specificity of the oxygenation represents a first limitation to overcome (Ajikumar et al 2010 ; Edgar et al 2016 ; Zhou et al 2015 ). Investigating the mechanism more in detail, earlier studies have suggested that promiscuous H-atom abstraction from both the isomeric olefinic precursors, taxa-4(5),11(2)-diene and taxa-4(20),11(2)-diene, forming an allylic radical followed by oxygen insertion can lead to the formation of T5OH (Hefner et al 1996 ; Jennewein et al 2004 ).…”

“…Investigating the mechanism more in detail, earlier studies have suggested that promiscuous H-atom abstraction from both the isomeric olefinic precursors, taxa-4(5),11(2)-diene and taxa-4(20),11(2)-diene, forming an allylic radical followed by oxygen insertion can lead to the formation of T5OH (Hefner et al 1996 ; Jennewein et al 2004 ). On the other hand, two independent recent studies have suggested an epoxide intermediate (Barton et al 2016 ; Edgar et al 2016 ) and formation of a mixture of products derived from taxa-4(5),11(2)-diene and a single oxidation product from taxa-4(20),11(2)-diene, concluding that an unstable epoxide intermediate may contribute to the native product profile of CYP725A4 (Edgar et al 2016 ). In contrast, and supporting promiscuity of CYP725A4, Biggs and co-workers performed in vivo characterization in engineered E. coli and in vitro characterization in a nanodisc platform, confirming the formation of T5OH, OCT, iso-OCT and additional oxygenated taxoids at titers exceeding 500 mg L −1 (Biggs et al 2016a ).…”

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

“…In 2014, Yadav studied a CYP725A4 construct and found that CYP725A4 acts on 4 substrates to form 12 products, and that taxadiene-5α-ol was only a very minor product [13]. 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].…”