Cytochrome p450 taxadiene 5alpha-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis.
The first oxygenation step in the biosynthesis of the anticancer drug taxol in Taxus species is the cytochrome p450-mediated hydroxylation (with double bond migration) of the diterpene olefin precursor taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol. A homology-based cloning strategy, employing an induced Taxus cell library, yielded a cDNA encoding taxadiene 5alpha-hydroxylase, which was functionally expressed in yeast and insect cells. The recombinant enzyme was characterized and shown to efficiently utilize both taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene (as an adventitious substrate) to synthesize taxa-4(20),11(12)-dien-5alpha-ol. This hydroxylase resembles, in sequence and properties, other cytochrome p450 oxygenases of taxol biosynthesis. The utilization of both taxadiene isomers in the formation of taxa-4(20),11(12)-dien-5alpha-ol is novel, suggesting a reaction mechanism involving promiscuous radical abstraction with selective oxygen insertion rather than epoxidation of the C4,C5-alkene of the natural substrate and allylic rearrangement of the resulting taxa-11(12)-en-4,5epoxide.
Jennewein
Stefan
S
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Long
Robert M
RM
Williams
Robert M
RM
Croteau
Rodney
R
D000475:Alkenes / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism; D017239:Paclitaxel / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D043823:Taxoids / Q000378:metabolism; D020946:Taxus / Q000201:enzymology
D000475:Alkenes; D004224:Diterpenes; D043823:Taxoids; C093125:taxa-4(5),11(12)diene; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D007535:Isomerases; D017239:Paclitaxel