Cytochrome P450 oxygenases of Taxol biosynthesis

Abstract

Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (>70%) but low similarity (<30%) to, and significant evolutionary distance from, other plant P450s. Despite their high similarity, functional analysis of these hydroxylases demonstrated distinctive substrate specificities responsible for an early bifurcation in the biosynthetic pathway after the initial hydroxylation of the taxane core at C5, leading into a biosynthetic network of competing, but interconnected, branches. The use of surrogate substrates, in cases where the predicted taxoid precursors were not available, led to the discovery of two core oxygenases, the 2α-and the 7β-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.

Terpenoid biomaterials

“…In contrast, terpenoid-modifying enzymes, including the P450s (e.g. Kaspera and Croteau, 2006 (Christianson, 2006;Starks et al, 1997;Wise and Croteau, 1999). By transient stabilization of these carbocations, TPS allows enzyme-specific isomerizations, various rearrangements, cyclizations, and eventually proton elimination or water termination to yield the many cyclic and acyclic terpenoid carbon skeletons found in plants.…”

Getting to the bottom of Taxol biosynthesis by fungi

“…(Baloglu and Kingston 1999;Itokawa 2003) but Taxol (if present at all) represents only a minor fraction of the total taxane complement. The biosynthesis of Taxol and other taxanes is well characterized Kaspera and Croteau 2006;Heinig and Jennewein 2009) and appears to follow an anastamosing pattern that yields several physiologically-active products as well as metabolic dead ends (Fig. 1).…”

Downstream reactions and engineering in the microbially reconstituted pathway for Taxol

“…For example, gene synthesis and chromosomal engineering methodology helped expedite the heterologous transfer and reconstitution process for artemisinin (an antimalarial compound) and Taxol intermediate production (Ajikumar et al 2010;Martin et al 2003;Ro et al 2006). The downstream pathways for complex isoprenoid compounds typically feature several steps catalyzed by plant cytochrome P450 enzymes and a partner reductase Jennewein et al 2005;Kaspera and Croteau 2006). Besides the usual challenges in establishing functional enzymatic activity in a heterologous host, these enzymes are membrane bound and may benefit from a heterologous cellular structure similar to that found in plants.…”

“…In the case of Taxol, there are reactions dedicated to hydroxylations, oxidation, epoxidation, acetylations, benzoylations, and an amino acid modification and addition. Notably, there is the utilization of several cytochrome P450 hydroxylase enzymes within the Taxol tailoring reactions (Jennewein et al 2004b;Kaspera and Croteau 2006).…”