CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane.
Taxa-4(5),11(12)-diene is the first committed precursor of functionalized taxanes such as paclitaxel, a successful anticancer drug. Biosynthesis of taxanes in yew involves several oxidations, a number of which have been shown to be catalyzed by cytochrome P-450 oxygenases. Hydroxylation of the C-5alpha of taxa-4(5),11(12)-diene is believed to be the first of these oxidations, and a gene encoding a taxa-4(5),11(12)-diene 5alpha-hydroxylase (CYP725A4) was recently described (Jennewein, S., Long, R. M., Williams, R. M., and Croteau, R. (2004) Chem. Biol. 11, 379-387). In an attempt to produce the early components of the paclitaxel pathway by a metabolic engineering approach, cDNAs encoding taxa-4(5),11(12)-diene synthase and CYP725A4 were introduced in Nicotiana sylvestris for specific expression in trichome cells. Their co-expression did not lead to the production of the expected 5alpha-hydroxytaxa-4(20),11(12)-diene. Instead, taxa-4(5),11(12)-diene was quantitatively converted to a novel taxane that was purified and characterized. Its structure was determined by NMR analysis and found to be that of 5(12)-oxa-3(11)-cyclotaxane (OCT) in which the eight-carbon B-ring from taxa-4(5),11(12)-diene is divided into two fused five-carbon rings. In addition, OCT contains an ether bridge linking C-5 and C-12 from opposite sides of the molecule. OCT was also the sole major product obtained after incubation of taxa-4(5),11(12)-diene with NADPH and microsomes prepared from recombinant yeast expressing CYP725A4. The rearrangement of the taxa-4(5),11(12)-diene ring system is thus mediated by CYP725A4 only and does not rely on additional enzymes or factors present in the plant. The complex structure of OCT led us to propose a reaction mechanism involving a sequence of events so far unknown in P-450 catalysis.
Rontein
Denis
D
Librophyt, Centre de Cadarache, 13115 St. Paul-Lez-Durance, France, Spectropole, Campus Scientifique de Saint Jérôme, Aix-Marseille Université, Marseille cedex 20, France. denis.rontein@librophyt.com
Onillon
Sandrine
S
Herbette
Gaëtan
G
Lesot
Agnes
A
Werck-Reichhart
Danièle
D
Sallaud
Christophe
C
Tissier
Alain
A
D000475:Alkenes / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000096:biosynthesis; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D006900:Hydroxylation; D007535:Isomerases / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D017239:Paclitaxel / Q000096:biosynthesis; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D043823:Taxoids / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
C529155:5(12)-oxa-3(11)-cyclotaxane; D000475:Alkenes; D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; D010940:Plant Proteins; D043823:Taxoids; C093125:taxa-4(5),11(12)diene; D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel