Taxadiene synthase

Transgenic tomato fruit expressing taxadiene synthase were able to reroute the production of taxadiene, allowing extraction of large amounts of highly purified taxadiene from freeze-dried tomato (Kovacs et al, 2007). 

HUANG, E. X., HUANG, Q. L WILDUNG, M. R CROTEAU, R SCOTT, A. I. Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the taxol biosynthetic pathway, Protein Expression Purification, 1998,13,90-%. 

WILLIAMS, D. C., WILDUNG, M. R., JIN, A. Q. W., DALAL, D., OLIVER, J. S., COATES, R. M., CROTEAU, R., Heterologous expression and characterization of a "pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction, Arch. Biochem. Biophys., 2000, 379, 137-146. 

Wildung, M.R. and Croteau, R. (1996) A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. /. Biol. Chem., 271, 9201-4. 

To date over 30 plant terpenoid synthases have been cloned as cDNAs, and many of these were found to encode enzymes of secondary metabolism (43). Isolation and analysis of six genomic clones encoding monoterpene ((—)-pinene and (—)-limonene), sesquiterpene ((E)-a-bisabolene and S-selinene) and diterpene (abietadiene) synthases from Abies grandis, and a diterpene (taxadiene) synthase from Taxus brevifolia have been reported (44). Overexpression of a cotton farnesyl diphosphate synthase (EPPS) in transgenic Artemesia annua has resulted in 3- to 4-fold increase in the yield of the sesquiterpenoid anti-malarial drug, artemisinin, in hairy roots (45). 

Williams, D. C., Barroll, B. J., Jin, Q., Rithner, C. D., Lenger, S. R., Floss, H. G., Coates, R. M.,Williams, R. M., Croteau, R., Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxa diene precursor of taxol catalyzed by recombinant taxadiene synthase,... 

A precursor to taxol, an anticancer drug, has also been produced from glucose in E. coli. IPP and DMAPP react together to form geranylgeranyl diphosphate which is transformed to taxadiene by taxadiene synthase. Enzymes for some of these reactions were obtained from species in the Taxus genus of yews. A combinatorial approach was employed, in which expression of pathway enzymes was varied to achieve final concentrations of taxadiene over 1 gl , a 15000-fold improvement over the control strain [40]. 

The genes for several of the key enzymes of this pathway have been cloned and expressed in Escherichia coli. This is the case for taxadiene synthase (560), taxadien-5a-ol-0-acetyltransferase (561), taxane 2a-0-benzoyltransferase (562) and 10-deacetylbaccatin III-10/ -O-acetyltrans-ferase (563). The gene for taxane 10/ -hydroxylase has been cloned and expressed in yeast (564). 

Wildung MR, Croteau R (1996) A cDNA Clone for Taxadiene Synthase, the Diterpene Cyclase That Catalyzes the Committed Step of Taxol Biosynthesis. J Biol Chem 271 9201... 

Williams DC, Carroll BJ, Jin Q, Rithner CD, Lenger SR, Floss HG, Coates RM, Williams RM, Croteau R (2000) Intramolecular Proton Transfer in the Cyclization of Geranylgeranyl Diphosphate to the Taxadiene Precursor of Taxol Catalyzed by Recombinant Taxadiene Synthase. Chem Biol 7 969... 

Figure 4.50 Proposed mechanism for the cyclization of geranylgeranyl diphosphate by taxadiene synthase.

The abietadiene synthase from fir was purified and internal amino-acid sequence information was employed to isolate the corresponding clone from a wound-induced stem cDNA library [147]. The cDNA encodes an 868 residue protein (99,536 Da) bearing a plastidial transit peptide and exhibiting the greatest sequence similarity to the only other known gymnosperm diterpene cyclase, taxadiene synthase from Taxus brevifolia [130]. Preliminary studies have identified a highly active truncation starting at V85 which approximates the preprotein-mature protein junction. 

The diterpene taxol from Pacific yew is one of the most powerful anticancer agents in therapeutic use today. In a series of seminal discoveries, the group of Croteau has dissected the pathway to this complex molecule at the enzymatic and molecular genetic levels, leading to recent success in partial reconstmction of the pathway in yeast [33]. The diterpene synthase taxadiene synthase catalyzes the cyclization of GGPP as the first committed step in taxol formation [34]. 

It uses a series of enzyme-controlled reactions. The step responsible for the creation of the unusual core is a cyclization reaction which changes geranylgeranyl diphosphate into taxa-4,11-diene. This then undergoes a variety of processes involving oxidation, acylation, and addition of a side chain to form taxol. The structure of the enzyme responsible for this, taxadiene synthase, was reported injanuary 2011. 

Figure 8.4 Paclitaxel biosynthetic pathway. TS taxadiene synthase TYHSa taxa-diene 5a-hydroxylase TAT taxadien-5a-ol 0-acetyl transferase TYHIO taxane 10 -hydroxylase TYH14 taxoid 14 -hydroxylase TBT taxane 2a-0-benzoyltransferase DBAT ...

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