Unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase

Yadav, Vikramaditya G.

doi:10.1016/j.molcatb.2014.10.004

Cited by 20 publications

(39 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this communication, we report the results of our own attempts to produce T5α-ol in E. coli by expressing TS and CYP725A4, along with upstream isoprenoid pathway genes, GGPPS, and P450 interacting proteins. Similar experiments have already been done previously by other groups [15–17, 22], but we deem it important to publish our independently generated data, especially considering the variable and confusing results reported in the literature. Our aim is to investigate the effect of redox partners on product formation and bring attention to the limitations of using TS and CYP725A4 in producing paclitaxel precursors (at least in E. coli ), which may not have been adequately addressed in previous publications, and stimulate more interdisciplinary studies into this bottleneck in paclitaxel metabolic engineering.…”

Section: Introductionsupporting

confidence: 53%

“…However, when heterologously expressed in E. coli , CYP725A4 has been shown to convert endotaxadiene mainly to 5(12)-oxa-3(11)-cyclotaxane (OCT) and 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) (Fig. 1) [15–17] in vivo , neither of which have been reported to occur naturally in yew, nor serve as precursors to paclitaxel.…”

Section: Introductionmentioning

confidence: 99%

“…taxadienes and T5α-ol), while CYP725A4 mainly generates undesired non-paclitaxel forming side products (i.e. OCT and iso-OCT) [15–17]. Thus, it still remains a challenge to find and optimize heterologous expression systems that can effectively use TS and CYP725A4 to produce high levels of T5α-ol [16, 21].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli

Sagwan-Barkdoll

Anterola

2017

Biotech and App Biochem

View full text Add to dashboard Cite

CYP725A4 is a P450 enzyme from Taxus cuspidata that catalyzes the formation of taxadiene-5α-ol (T5α-ol) from taxadiene in paclitaxel biosynthesis. Past attempts expressing CYP725A4 in heterologous hosts reported the formation of 5(12)-oxa-3(11)-cyclotaxane (OCT) and/or 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) instead of, or in addition to, T5α-ol. Here, we report that T5α-ol is produced as a minor product by Escherichia coli expressing both taxadiene synthase and CYP725A4. The major products were OCT and iso-OCT, while trace amounts of unidentified monooxygenated taxanes were also detected by gas chromatography-mass spectrometry. Since OCT and iso-OCT had not been found in nature, we tested the hypothesis that protein-protein interaction of CYP725A4 with redox partners, such as cytochrome P450 reductase (CPR) and cytochrome b5, may affect the products formed by CYP725A4, possibly favoring the formation of T5α-ol over OCT and iso-OCT. Our results show that coexpression of CYP725A4 with CPR from different organisms did not change the relative ratios of OCT, iso-OCT, and T5α-ol, while cytochrome b5 decreased overall levels of the products formed. Although unsuccessful in finding conditions that promote T5α-ol formation over other products, we used our results to clarify conflicting claims in the literature and discuss other possible approaches to produce paclitaxel via metabolic and enzyme engineering.

show abstract

Section: Introductionsupporting

confidence: 53%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli

Sagwan-Barkdoll

Anterola

2017

Biotech and App Biochem

View full text Add to dashboard Cite

show abstract

“…In contrast to bacterial class I and III hydroxylases, eukaryotic class II P450s are membrane associated that are characterized by a low solubility when functional reconstitution is carried out in conventional bacterial hosts such as Escherichia coli [ 10 ]. Moreover, plant-derived class II P450s often yield complex product mixtures [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

et al. 2016

View full text Add to dashboard Cite

BackgroundDe novo production of multi-hydroxylated diterpenoids is challenging due to the lack of efficient redox systems.ResultsIn this study a new reductase/ferredoxin system from Streptomyces afghaniensis (AfR·Afx) was identified, which allowed the Escherichia coli-based production of the trihydroxylated diterpene cyclooctatin, a potent inhibitor of human lysophospholipase. This production system provides a 43-fold increase in cyclooctatin yield (15 mg/L) compared to the native producer. AfR·Afx is superior in activating the cylcooctatin-specific class I P450s CotB3/CotB4 compared to the conventional Pseudomonas putida derived PdR·Pdx model. To enhance the activity of the PdR·Pdx system, the molecular basis for these activity differences, was examined by molecular engineering.ConclusionWe demonstrate that redox system engineering can boost and harmonize the catalytic efficiency of class I hydroxylase enzyme cascades. Enhancing CotB3/CotB4 activities also provided for identification of CotB3 substrate promiscuity and sinularcasbane D production, a functionalized diterpenoid originally isolated from the soft coral Sinularia sp.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0487-6) contains supplementary material, which is available to authorized users.

show abstract

“…As part of the same study, CYP725A4 was expressed in yeast, and microsomes were prepared that also converted taxadiene to OCT rather than taxadiene-5α-ol [12]. 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].…”

Section: Introductionmentioning

confidence: 99%

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

Rouck

Biggs

Kambalyal

et al. 2017

Protein Expression and Purification

View full text Add to dashboard Cite

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 KD of 2.1 μM ± 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 Vmax of 30 ± 8 pmol/min/nmolCYP725A4 and a KM of 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.

show abstract

Unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase

Cited by 20 publications

References 28 publications

Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli

Taxadiene‐5α‐ol is a minor product of CYP725A4 when expressed in Escherichia coli

Identification, characterization and molecular adaptation of class I redox systems for the production of hydroxylated diterpenoids

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

Contact Info

Product

Resources

About