Molecular cloning and characterization of a cytochrome P450 taxoid 2α-hydroxylase involved in Taxol biosynthesis

Chau, MyDoanh; Croteau, Rodney

doi:10.1016/j.abb.2004.04.016

Cited by 90 publications

(67 citation statements)

References 41 publications

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“…A cladogram of the taxoid oxygenases was constructed with rooting at the C5a-hydroxylase (Jennewein et al, 2004b), and the pattern was consistent with the preliminary hydroxylation steps leading from C5 hydroxylation to C10 or C13 hydroxylation and with the early emergence of C14 hydroxylation after C5 hydroxylation as a major side route. Phylogenic considerations placed the taxoid C7 hydroxylase closer to the C5 hydroxylase than might have been anticipated based on the proposed sequence of hydroxylations deduced from the relative abundances of taxoid metabolites functionalized at the various positions (Floss and Mocek, 1995) but in agreement with recent experimental implications for the relative placement of the 7b-hydroxylase (Chau and Croteau, 2004;Chau et al, 2004a). With at least two pathway oxygenases still unaccounted for, and many of the cytochrome P450 genes in the group of still uncertain function, this preliminary phylogeny of pathway steps can only be regarded as approximate.…”

Section: Cytochrome P450 Taxoid Oxygenasessupporting

confidence: 74%

“…Nevertheless, the natural occurrence in Taxus of a broad range of taxusin-like metabolites bearing additional oxygen functional groups at C1, C2 or C7 (Baloglu and Kingston, 1999) encouraged the use of this material as an alternate substrate for testing microsomal oxygenase activities and for screening cytochrome P450 clones in yeast. This approach led to the acquisition and characterization of the seemingly regioselective taxoid2a-hydroxylase (Chau and Croteau, 2004) and taxoid-7b-hydroxylase (Chau et al, 2004a).…”

Section: Cytochrome P450 Taxoid Oxygenasesmentioning

confidence: 99%

“…Conversions of the surrogate substrate (+)-taxusin mediated by taxoid 2a-hydroxylase and taxoid 7b-hydroxylase. 2a,7b-dihydroxy taxusin) ( Figure 6) indicated that these hydroxylases are capable of operating sequentially in the possible order of 7b-hydroxylation followed by 2a-hydroxylation on the way to the level of a taxadien-2a, 5a, 7b, 9a, 10b; 13a-hexaol at some level of acylation (Chau and Croteau, 2004).…”

Section: Cytochrome P450 Taxoid Oxygenasesmentioning

confidence: 99%

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Taxol Biosynthesis and Molecular Genetics

et al. 2006

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Biosynthesis of the anticancer drug Taxol in Taxus (yew) species involves 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methyl erythritol phosphate pathway for isoprenoid precursor supply. Following the committed cyclization to the taxane skeleton, eight cytochrome P450-mediated oxygenations, three CoA-dependent acyl/aroyl transfers, an oxidation at C9, and oxetane (D-ring) formation yield the intermediate baccatin III, to which the functionally important C13-side chain is appended in five additional steps. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug, and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, Taxus suspension cells (induced for taxoid biosynthesis by methyl jasmonate) were used for feeding studies, as the foundation for cell-free enzymology and as the source of transcripts for cDNA library construction and a variety of cloning strategies. This approach has led to the elucidation of early and late pathway segments, the isolation and characterization of over half of the pathway enzymes and their corresponding genes, and the identification of candidate cDNAs for the remaining pathway steps, and it has provided many promising targets for genetically engineering more efficient biosynthetic production of Taxol and its precursors.

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Section: Cytochrome P450 Taxoid Oxygenasessupporting

confidence: 74%

Section: Cytochrome P450 Taxoid Oxygenasesmentioning

confidence: 99%

Section: Cytochrome P450 Taxoid Oxygenasesmentioning

confidence: 99%

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Taxol Biosynthesis and Molecular Genetics

et al. 2006

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“…Proposed mechanism for cytochrome P450-mediated conversion of taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene to taxa-4(20),11(12)-dien-5α-ol involving hydrogen abstraction at C5 or C20 to provide the common allylic radical, followed by oxygen insertion at the C5α-face. Taxa-4(5),11(12)-diene and taxa-4(20),11(12)-diene isomerization was not observed (Jennewein et al 2004) Early bifurcation of the Taxol biosynthetic pathway following the 5α-hydroxylation step, and involving the taxoid 13α-(T13OH), 10β-(T10OH), 14β-(T14OH) and presumptive 9α-(T9OH) hydroxylases. The broken arrows indicate subsequent, but as yet undefined, metabolic steps…”

Section: Resultsmentioning

confidence: 94%

Cytochrome P450 oxygenases of Taxol biosynthesis

2006

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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.

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“…For example, the biosynthesis of taxol, a diterpenoid having multiple anticancer properties, consists of approximately 20 steps, at least nine of which are thought to be cytochrome P450-mediated oxygenations [13]. Sesquiterpenes represent the largest subfamily of natural terpenoids, and P450s are assumed to play a major role in their biosynthetic pathways.…”

Section: Introductionmentioning

confidence: 99%

Zingiber zerumbet CYP71BA1 catalyzes the conversion of α-humulene to 8-hydroxy-α-humulene in zerumbone biosynthesis

Okamoto

Harada

et al. 2010

Cell. Mol. Life Sci.

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Plant cytochrome P450s are involved in the biosynthesis of various classes of secondary metabolites. To elucidate the biosynthesis of zerumbone, a sesquiterpenoid with multiple potential anticancer properties, a family of P450 genes expressed in rhizomes of Zingiber zerumbet Smith, were cloned using a PCR-based cloning strategy. After functional expression in yeast, one of these P450s was found to convert α-humulene into 8-hydroxy-α-humulene, a proposed intermediate of zerumbone biosynthesis. This P450 has been designated CYP71BA1, a new member of the CYP71 family. CYP71BA1 transcripts were detected almost exclusively in rhizomes and showed a similar expression pattern to ZSS1 transcripts during rhizome development. Coexpression of a gene cluster encoding four enzymes of the mevalonate pathway with CYP71BA1 and ZSS1 in Escherichia coli leads to the production of 8-hydroxy-α-humulene in the presence of mevalonate, suggesting the possibility of microbial production of this zerumbone intermediate from a relatively simple carbon source by metabolic engineering.

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Molecular cloning and characterization of a cytochrome P450 taxoid 2α-hydroxylase involved in Taxol biosynthesis

Cited by 90 publications

References 41 publications

Taxol Biosynthesis and Molecular Genetics

Taxol Biosynthesis and Molecular Genetics

Cytochrome P450 oxygenases of Taxol biosynthesis

Zingiber zerumbet CYP71BA1 catalyzes the conversion of α-humulene to 8-hydroxy-α-humulene in zerumbone biosynthesis

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