Taxoid-hydroxylases

Figure 4.52 Reactions catalyzed by the taxoid lOp-hydroxylase, taxoid 13a-hydroxylase, and taxoid 14p-hydroxylase. AU of these hydroxylases accept either the 5a-alcohol or the corresponding acetate ester as alternate substrates. Only the most favorable routes are illustrated.

Beside the above-mentioned hydroxylation steps, oxygenation at C-9 is also presumed to occur fairly early in the pathway. A clone for the cytochrome P450 taxoid 9a-hydroxylase was tentatively identified, but testing the 5a,13a-diol, the 5a,10p-diol, and the corresponding 5a-acetates as substrates could not provide sufficient biosynthetic products to permit NMR-based confirmation of structures [251]. 

Intermediate oxygenation steps of the pathway from the level of a taxadien tetraol onward have been more difficult to approach since the intermediates are not known or, if predicted, are not available for testing. Therefore, this mid-seetion of the pathway is not well defined in the reaction order. Such difficulties impose the use of accessible surrogate substrates to explore such hydroxylations. For this purpose, taxusin, the tetraacetate of taxa-4(20),l l(12)-dien-5a,9a,10p,13a-tetraol, was used as a surrogate substrate to functionally evaluate the cytochrome P450 clones for taxoid C-1, C-2, and C-1 hydroxylase activities, and for the presumed C-4, C-20-epoxidase. 

Figure 4.53 Conversions of the surrogate substrate (-i-)-taxusm to 2a,7p-dihydroxytaxusin mediated by taxoid 2a-hydroxylase and taxoid 7p-hydroxylase.

On the way to baccatin III, there are several more steps. Two oxygenation reactions remain to be defined, and the corresponding gene are missing. These are the presumed cytochrome P450-mediated taxoid C-ip-hydroxylase and the C-4p,C-20-epoxidase, leading to the oxetane formation the oxidation of the C-9a-hydroxyl function, likely mediated by a cytochrome P450 hydroxylase and the transfer of three ester functions C-2 benzoate, C-4 acetate, and C-10 acetate. 

Taxoid metabolism taxoid 14P-hydroxylase is a cytochrome P450-dependent monooxygenase. Arch Biochem Biophys 413 262-270... 

Chau M, Croteau R (2004) Molecular cloning and characterization of a cytochrome P450 taxoid 2a-hydroxylase involved in Taxol biosynthesis. Arch Biochem Biophys 427 48-57... 

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 ...

Knockout of taxoid 14 -hydroxylase (T140H) [78] Side-route pathway enzyme T. media cell suspension Decrease in the three byproducts of taxanes no report about paclitaxel level... 

A. et al. (2011) Antisense-induced suppression of taxoid 14 beta-hydroxylase gene expression in transgenic Taxus... 

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