Taxol Biosynthesis and Molecular Genetics

Croteau, Rodney; Ketchum, Raymond E.B.; Long, Robert M.; Kaspera, Rüdiger; Wildung, Mark R.

doi:10.1007/s11101-005-3748-2

Cited by 410 publications

(358 citation statements)

References 99 publications

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“…Benzoyl-CoA, and [7][8][9][10][11][12][13][14] C]benzoyl-CoA (1.1 mCi/mol) employed for preliminary kinetic assay development, were prepared as previously described, as were tigloyl-CoA (from tiglic acid) and N-debenzoyl-2′-deoxytaxol (5) [17,19].…”

Section: Substrates and Reagentsmentioning

confidence: 99%

“…A 100-µL aliquot of protein solution (up to 100 µM TAX10 protein) in the above noted buffer was combined with up to 500 µM of either N-debenzoyl-2′-deoxytaxol (5) or N-debenzoyltaxol (6) and up to 500 µM [7][8][9][10][11][12][13][14] C]benzoyl-CoA in the same buffer, and incubated at 31°C for up to 1 h. Assay mixtures were then extracted with two 0.1-mL portions of CH 2 Cl 2 , and the organic extracts were pooled and reduced to dryness under a stream of purified N 2 . The residue was dissolved in 50 µL of CH 3 CN, filtered through a 0.2-µm nylon syringe filter (Millex-GN, Millipore, Bedford, MA) into a sample vial, and analyzed by HPLC with UV-and radiomonitoring (Agilent 1100 Series with G1311A quaternary pump, and coupled to a G1314A UV-detector and Packard Series A-100 radio-detector).…”

Section: Enzyme Preparation and Assaymentioning

confidence: 99%

“…This parental olefin is then functionalized by a series of oxygenations, acylations, and other reactions to yield the late stage intermediate baccatin III (4, Fig. 1) [14], upon which the functionally significant Nbenzoyl phenylisoserine side chain [15] is then assembled at the C13-O-position to complete the pathway to Taxol (7).…”

mentioning

confidence: 99%

“…Feeding studies with Taxus tissues, using both amino acid precursors and advanced taxoid intermediates, metabolite profiling of Taxus cells in culture, and demonstration of relevant activities in cell free enzyme systems (see [14,16,17] for reviews of this work) have indicated that side chain construction requires five steps ( Fig. 1) involving aminomutase-catalyzed conversion of (S)-α-phenylalanine (1) to (R)-β-phenylalanine (2), ligation to the corresponding CoA ester (3), transfer of the aminoacyl group to the C13-hydroxyl of baccatin III (4) to yield N-debenzoyl-2′-deoxytaxol (i.e., 13-O-β-phenylalanoyl baccatin III, 5), hydroxylation at the 2′-position in the side chain to afford N-debenzoyltaxol (i.e., 13-O-phenylisoserinoyl baccatin III, 6), and N-benzoylation as the final side chain modification.…”

mentioning

confidence: 99%

“…In the case of the transfer reactions, functional screening of a family of previously acquired Taxus acyl/aroyl transferase cDNAs [13] yielded the C13 phenylpropanoid side chain-CoA acyl transferase that mediates the 13-O-esterification of baccatin III [20], and the benzoyl CoA-dependent transferase responsible for the final benzamidation step of the Taxol pathway [19]. The lack of absolute substrate specificity by the recombinant transferases led to some ambiguities with regard to the precise timing of hydroxylation and acyl transfer steps in side chain assembly [14] that were only recently resolved by demonstration that the cytochrome P450 2′-hydroxylase operates only on Ndebenzoyl-2′-deoxytaxol (5), thus implicating the construction sequence of O-esterification, followed by 2′-hydroxylation, followed by N-benzamidation [17]. In this context, it is important to note that the recombinant terminal N-transferase was characterized using the more readily available N-debenzoyl-2′-deoxytaxol (5) as a surrogate taxoid co-substrate [19].…”

mentioning

confidence: 99%

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Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

Long

Lagisetti

Coates

et al. 2008

Archives of Biochemistry and Biophysics

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The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of β-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2′-deoxytaxol, followed by hydroxylation on the side chain at the C2′-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2′-deoxytaxol [Walker et al., Proc. Natl. Acad. Sci. USA 99 (2002) 9166-9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-β-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2′-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2′-hydroxylation prior to N-benzoylation. Selectivity for the acyl/ aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield taxol C or taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.

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Section: Substrates and Reagentsmentioning

confidence: 99%

Section: Enzyme Preparation and Assaymentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

Long

Lagisetti

Coates

et al. 2008

Archives of Biochemistry and Biophysics

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Phenylpropanoid Metabolism

Rock

2017

Encyclopedia of Life Sciences

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Phenylpropanoid compounds encompass a wide range of structural classes with diverse biological functions in defence, survival and structural support associated with normal plant development (belying the term 'secondary metabolite'). The biosynthesis of phenylpropanoids is regulated by diverse environmental stimuli. Phenylpropanoid metabolism (other than flavonoids, not covered here) occupies a central place in the general aromatic metabolism of plants from shikimate to phenylalanine to lignin polymers and also to coumarins, phenolic volatiles and hydrolysable tannins. In recent years, genetics and biochemistry, along with methodology‐driven, computational, transgenic and comparative transcriptomic approaches, have led to significant advances in the identification of the families of genes encoding enzymes, transporters, regulatory factors involved in phenylpropanoid metabolism and a clearer picture of their functions in biotic and abiotic stress responses, plant development and enzyme/pathway evolution driven by interactions of species with their environment. Key Concepts Having one phenyl aromatic ring with one or more hydroxyl groups attached gives phenylpropanoids amphiphilic and reducing properties, underlying their ability to physically interact with other biomolecules. Plants invest a large percentage of their fixed carbon into synthesising phenylpropanoids, from simple volatile phenolic acids such as the defence hormone salicylic acid to complex polyphenolic flavonoids encompassing thousands of compounds with myriad physiological and adaptive functions. The shikimate pathway is the starting point for phenylpropanoid biosynthesis from shikimate product phenylalanine ( l ‐Phe), via the intermediate chorismate, which also serves as substrate for the synthesis of quinones and tocopherols important as electron acceptors in photosynthesis and aerobic respiration. Phenylpropanoid biosynthesis proceeds from deamination of Phe to cinnamate by the rate‐limiting and environmentally regulated enzyme PAL, followed by oxidation of the ring to p ‐coumarate, its activation with coenzymeA and a series of hydroxylation, methylation and reduction reactions to give cinnamic acids, ‐aldehydes and ‐alcohols that serve as substrates to generate a wide range of complex structures, notably polymers of coumaroyl, conferyl and sinapyl alcohols comprising lignin. Recent studies have established the major pathway in plants which proceeds from p ‐coumaroyl‐CoA → p ‐coumaryl‐shikimate → caffeoyl‐shikimate → caffeoyl‐CoA → feruloyl CoA → coniferaldehyde → 5‐OH coniferaldehyde → sinapaldehyde → sinapate/sinapyl alcohol (oxidation versus reduction, respectively), instead of the original model of a grid/matrix of parallel ring oxidation and side‐chain redox pathways from hydroxycinnamic acids to alcohols. Synthesis involves three subcellular compartments: chloroplast for Phe, cytosol for sinapoyl‐esters and the vacuole for trans‐esterifications. The identity of specific transporters, temporal‐ or organ‐specific regulatory factors for phenylpropanoid flux to (in)soluble polymers in the apoplast in response to biotic and abiotic stressors and whether lignin formation proceeds via precise channeling of individual precursors through metabolons (temporary structural–functional complexes formed between sequential enzymes) are key questions of practical significance that remain to be answered.

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Plant Terpenoids

Keeling¹,

Bohlmann²

2008

Wiley Encyclopedia of Chemical Biology

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Terpenoids are the largest class of all known natural products. Plants produce a variety of terpenoid compounds that number in the thousands. Some terpenoids are involved in plant growth and development directly (i.e., in primary metabolism), but most plant terpenoids are thought to function in interactions of plants with their biotic and abiotic environment and have traditionally been referred to as secondary metabolites. In addition to the isolation and identification of plant terpenoids, research has concentrated on the biosynthesis, the biological function, and the exploitation of plant terpenoids for human use as biomaterials and pharmaceuticals. Plant terpenoids are biosynthesized from C 5 precursors by the action of prenyl transferases and terpenoid synthases. Often, terpenes are acted on by cytochromes P450 and other enzymes to increase their functionalization. Terpenoid biosynthesis in plants involves several subcellular compartments. The accumulation of terpenoids requires efficient transport systems and specialized anatomical structures. Using isoprene (a hemiterpene), menthol (a monoterpene), artemisinin (a sesquiterpene), and paclitaxel [better known under the registered trademark Taxol (Bristol Myers Squibb, New York)] and diterpene resin acids (diterpenes) as examples, we highlight some strategies, techniques, and results of plant terpenoid research with a strict focus on the low‐molecular‐weight (C 5 –C 20 ) terpenoids of specialized plant metabolism.

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Taxol Biosynthesis and Molecular Genetics

Cited by 410 publications

References 99 publications

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

Phenylpropanoid Metabolism

Plant Terpenoids

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