Cyclization of Geranylgeranyl Diphosphate to Taxa-4(5),11(12)-diene Is the Committed Step of Taxol Biosynthesis in Pacific Yew

Abstract

The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol.

Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway

“…In the case of [20-3 H 3 ] taxadiene (3), an incorporation rate of 1.1% into radiochemically pure Taxol (1) of T. brevifolia stem sections was previously reported (Koepp et al, 1995). The incorporation rate of this early precursor into chromatographically pure Taxol (1) in 7-day uninduced T. media cells in the present case was 2.9% (corrected for loss of one 3 H atom from C20 in the process).…”

“…The initial feeding study was carried out with taxa-4(20),11(12)-diene (3), the product of the committed pathway step and the presumptive parent of all taxoids (Koepp et al, 1995). This [20-3 H 3 ]-labeled precursor was incorporated into all taxoids in proportional quantities reflecting their approximate relative abundances in the cell extract (Figure 2).…”

“…The committed step in the biosynthesis of Taxol (1) and other taxoids is the cyclization of the universal diterpenoid precursor geranylgeranyl diphosphate (2) to taxa-4(20),11(12)-diene (3) to establish the taxane ring system (Koepp et al, 1995). The cyclization is then followed by the first hydroxylation of the pathway at the C5α-position, with allylic migration of the double bond, to afford the second established intermediate 5α-hydroxytaxa-4(20),11(12)-diene (4) (Figure 1) (Hefner et al, 1996;Jennewein et al, 2004).…”

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

“…Early efforts to identify the exact biosynthetic steps towards Taxol's production were published in the mid to late 1990s (Hefner et al 1998(Hefner et al , 1996Hezari et al 1995;Koepp et al 1995;Walker et al 1999;Wildung and Croteau 1996). This work identified the geranylgeranyl diphosphate synthase, taxadiene synthase, cytochrome P450 hydroxylase, and acetyltransferase needed to make the hypothesized first three dedicated intermediates of the Taxol pathway.…”

“…Considering that the current state-of-the-art is the production of early stage Taxol intermediates through heterologous hosts, this discussion will summarize success to date and the steps needed to realize and capitalize upon full pathway reconstitution. Hefner et al 1998), taxadiene synthase (Wildung and Croteau 1996;Koepp et al 1995;Huang et al 2001), taxadiene 5α-hydroxylase (Jennewein et al 2004a), taxane 2α-Obenzoyltransferase (TBT; Walker and Croteau 2000b), 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT; Walker and Croteau 2000a), baccatin III:3-amino-3-phenylpropanoyltransferase (BAPT; Walker et al 2002a), and phenylalanine aminomutase (PAM; Walker et al 2004). Other enzymes (taxadien-5α-ol-O-acetyl transferase, taxoid 10β-hydroxylase, taxoid 2α-hydroxylase, taxoid 9α-hydroxylase, taxane 13α-hydroxylase, taxoid 7β-hydroxylase, and 3′-N-debenzoyl-2′-deoxytaxol N-benzyoyltransferase) have been identified and/or characterized but not conclusively assigned a location in the overall pathway order (Walker et al 2000;Schoendorf et al 2001;Croteau et al 2006;Jennewein et al 2001;Walker et al 2002b).…”