10-Deacetylbaccatin

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Gabriele Fontana - One of the best experts on this subject based on the ideXlab platform.

  • novel c seco taxoids possessing high potency against paclitaxel resistant cancer cell lines overexpressing class iii β tubulin
    Bioorganic & Medicinal Chemistry Letters, 2009
    Co-Authors: Antonella Pepe, Gabriele Fontana, E Bombardelli, Liang Sun, Ilaria Zanardi, Cristiano Ferlini, Iwao Ojima
    Abstract:

    Novel C-seco-taxoids were synthesized from 10-Deacetylbaccatin III and their potencies evaluated against drug-sensitive and drug-resistant cancer cell lines. The drug-resistant cell lines include ovarian cancer cell lines resistant to cisplatin, topotecan, adriamycin and paclitaxel overexpressing class III β-tubulin, A2780TC1 and A2780TC3. The last two cell lines were selected through chronic exposure of A2780wt to paclitaxel and Pgp blocker cyclosporine. All novel C-seco-taxoids exhibited remarkable potency against A2780TC1 and A2780TC3 cell lines, and no cross resistance to cisplatin- and topotecan-resistant cell lines, A2780CIS and A2780TOP. Four of those C-seco-taxoids exhibit much higher activities than IDN5390 against paclitaxel-resistant cell lines, A2780ADR, A2780TC1 and A2780TC3. SB-CST-10202 possesses the best all-round high potencies across different drug-resistant cell lines. Molecular modeling studies, including molecular dynamics simulations, on the drug-protein complexes of class I and III β-tubulins were performed to identify possible cause of the remarkable potency of these C-seco-taxoids against paclitaxel-resistant cell lines overexpressing class III β-tubulin.

  • pharmacokinetics and metabolism in mice of idn 5390 13 n boc 3 i butylisoserinoyl c 7 8 seco 10 deacetylbaccatin iii a new oral c seco taxane derivative with antiangiogenic property effective on paclitaxel resistant tumors
    Drug Metabolism and Disposition, 2006
    Co-Authors: Roberta Frapolli, Elena Marangon, Marco Zaffaroni, Tina Colombo, Cristiano Falcioni, Renzo Bagnati, Carla Manzotti, Matteo De Simone, Maurizio Dincalci, Gabriele Fontana
    Abstract:

    IDN 5390 (13-(N-Boc-3-i-butylisoserinoyl)-C-7,8-seco-10-Deacetylbaccatin III) is a new taxane, derived from 7,8-C-seco-10-Deacetylbaccatin, selected for its ability to inhibit angiogenesis, mainly by acting on endothelial cell motility, and for its selective activity on class III beta-tubulin. In vivo, IDN 5390 shows activity against paclitaxel-sensitive and -resistant tumors when administered on a prolonged, continuous dosage schedule. We studied the pharmacokinetics and bioavailabilty of the drug in mice after single and repeated oral treatment. IDN 5390 was rapidly absorbed after oral administration, with good bioavailability (43%). After intravenous injection, it was extensively distributed in tissue, mainly the liver, kidney, and heart, with low but persistent levels in brain. The kinetics appear dose-dependent with a clearance of 2.6, 1.4, and 0.9 l/kg at, respectively, 60, 90, and 120 mg/kg, and a half-life 24, 36, and 54 min. After prolonged daily oral doses given for 2 weeks, we found that there was a decrease in drug availability; i.e., the area under the concentration-time curve value after p.o. daily administration on day 14 was 2-fold lower than that on day 1. Metabolism plays a major role in elimination of the drug, and at least 12 metabolites were identified in feces and urine. The percentage excreted as metabolites after an oral dose (42%) was higher than that after the i.v. dose (33%), suggesting a first-pass effect. Four metabolites were found in plasma at detectable levels; one of them, with restored taxane scaffold, is a species 3 times more potent than IDN 5390, possibly contributing to the observed anti-tumor activity.

  • Microbial transformation of 10-Deacetylbaccatin III (10-DAB) by Curvularia lunata and Trametes hirsuta
    Journal of Molecular Catalysis B-enzymatic, 2006
    Co-Authors: Alberto Arnone, Stefano Alemani, Adriana Bava, Gianluca Nasini, E Bombardelli, Gabriele Fontana
    Abstract:

    Abstract The microbial transformation of 10-Deacetylbaccatin III (10-DAB) ( 1a ) and 13-DeBAC ( 4b ) was investigated. Trametes hirsuta induced 13-oxidation of 10-DAB to give ( 4a ) in high yield, whereas incubation with Curvularia lunata resulted in the isolation of the 7- epi -10-DAB ( 2 ) and the 7- epi -10-oxo-10-DAB ( 3 ). 13-DeBAC ( 4b ) was biotransformed into compounds ( 4a ) and ( 4c ) by Alternaria alternata .

Torsten Binscheck - One of the best experts on this subject based on the ideXlab platform.

  • Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-Deacetylbaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
    2016
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten Binscheck
    Abstract:

    This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid–liquid extrac-tion under alkaline conditions followed by LC–MS–MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 3 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-Deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohy-droxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantita-tive evaluation revealed taxoid concentrations ranging from 4.5 to 132 mg/L (stomach content), 1 to 200 mg/L (urine), <0.5 to 12 mg/ L (cardiac blood), <0.5 to 7.3 mg/L (femoral blood), and 4.9 to 290 mg/L (bile). In brain tissue, none of these taxoids could be detected (<0.5 mg/L). In urine, after enzymatic hydrolysis, the con-centration of 3,5-dimethoxyphenol (3,5-DMP) was 23,000 mg/L. The alkaloidal diterpenoids were found in all postmortem samples. The newly developed LC–MS–MS method enables the identifica-tion of alkaloidal and non-alkaloidal diterpenoids and 3,5-dimethox-yphenol in human body fluids and tissues for the confirmation of accidental or intentional poisonings with yew plant material

  • eight cases of fatal and non fatal poisoning with taxus baccata
    Forensic Science International, 2013
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Norbert Felgenhauer, Benno Riesselmann, Sonja Roscher, Torsten Binscheck
    Abstract:

    This paper describes two fatalities, three non-fatal intentional and three accidental oral ingestions of yew (Taxus baccata) leaves. In all cases the post-mortem external examinations showed no signs of violence. Internal examinations revealed small green, needle-like particles on the tongue, in the esophagus and in the stomach. Yew leaves were also identified in the stomach contents, whereas Taxus leaves were cut into small pieces and then ingested in one case. The analytical method used was based on a liquid-liquid-extraction under alkaline conditions followed by LC-MS/MS analysis (QTRAP 5500). Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100×3) mm. The analytical method allows the simultaneous identification and quantification of the commercially available yew alkaloids taxoids (m/z): paclitaxel (854.2→105.0/286.1), 10-deacetyltaxol (10-DAT: 812.2→105.0/286.1), baccatin III (BAC III: 604.0→105.0/327.0), 10-Deacetylbaccatin III (10-DAB III: 562.1→105.0/327.0), cephalomannine [taxol B] (562.1→105.0/327.0) and of 3,5-dimethoxyphenol (3,5-DMP: 155.0→111.9/122.9) also encompassing the qualitative analysis of the alkaloidal diterpenoids (Q1→194.0/107.0); reference mass spectra obtained from a yew leaves extract: monoacetyltaxine (MAT: 568.4), taxine B (584.2), monohydroxydiacetyltaxine (MHDAT: 626.4), triacetyltaxine (TAT: 652.4), monohydroxytriacetyltaxine (MHTAT: 668.4). In both fatalities, paclitaxel, 10-DAT and cephalomannine were not identified in urine, cardiac and femoral blood but all taxoids and 3,5-DMP were present in stomach content and excreted into the bile. In urine, highest 3,5-DMP concentration was 7500 μg/L and 23,000 μg/L after enzymatic hydrolysis, respectively. In intentional and accidental poisonings, when electrocardiogram (ECG) examinations revealed ventricular tachycardia and/or prolonged QRS intervals, taxines were identified in plasma/serum, even after the ingestion of a few number of yew leaves, when 3,5-dimethoxyphenol was not even found. According to the data from one near-fatal intentional poisoning, elimination half-life of MAT, TAXIN B, MHDAT and MHTAT in serum was calculated with 11-13 h and taxines were detected up to t=+122 h post-ingestion of approximately two handfuls of yew leaves.

  • Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-Deacetylbaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
    Journal of Analytical Toxicology, 2012
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten Binscheck
    Abstract:

    This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid-liquid extraction under alkaline conditions followed by LC-MS-MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 × 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-Deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohydroxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantitative evaluation revealed taxoid concentrations ranging from 4.5 to 132 µg/L (stomach content), 1 to 200 µg/L (urine), Language: en

Thomas Grobosch - One of the best experts on this subject based on the ideXlab platform.

  • Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-Deacetylbaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
    2016
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten Binscheck
    Abstract:

    This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid–liquid extrac-tion under alkaline conditions followed by LC–MS–MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 3 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-Deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohy-droxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantita-tive evaluation revealed taxoid concentrations ranging from 4.5 to 132 mg/L (stomach content), 1 to 200 mg/L (urine), <0.5 to 12 mg/ L (cardiac blood), <0.5 to 7.3 mg/L (femoral blood), and 4.9 to 290 mg/L (bile). In brain tissue, none of these taxoids could be detected (<0.5 mg/L). In urine, after enzymatic hydrolysis, the con-centration of 3,5-dimethoxyphenol (3,5-DMP) was 23,000 mg/L. The alkaloidal diterpenoids were found in all postmortem samples. The newly developed LC–MS–MS method enables the identifica-tion of alkaloidal and non-alkaloidal diterpenoids and 3,5-dimethox-yphenol in human body fluids and tissues for the confirmation of accidental or intentional poisonings with yew plant material

  • eight cases of fatal and non fatal poisoning with taxus baccata
    Forensic Science International, 2013
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Norbert Felgenhauer, Benno Riesselmann, Sonja Roscher, Torsten Binscheck
    Abstract:

    This paper describes two fatalities, three non-fatal intentional and three accidental oral ingestions of yew (Taxus baccata) leaves. In all cases the post-mortem external examinations showed no signs of violence. Internal examinations revealed small green, needle-like particles on the tongue, in the esophagus and in the stomach. Yew leaves were also identified in the stomach contents, whereas Taxus leaves were cut into small pieces and then ingested in one case. The analytical method used was based on a liquid-liquid-extraction under alkaline conditions followed by LC-MS/MS analysis (QTRAP 5500). Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100×3) mm. The analytical method allows the simultaneous identification and quantification of the commercially available yew alkaloids taxoids (m/z): paclitaxel (854.2→105.0/286.1), 10-deacetyltaxol (10-DAT: 812.2→105.0/286.1), baccatin III (BAC III: 604.0→105.0/327.0), 10-Deacetylbaccatin III (10-DAB III: 562.1→105.0/327.0), cephalomannine [taxol B] (562.1→105.0/327.0) and of 3,5-dimethoxyphenol (3,5-DMP: 155.0→111.9/122.9) also encompassing the qualitative analysis of the alkaloidal diterpenoids (Q1→194.0/107.0); reference mass spectra obtained from a yew leaves extract: monoacetyltaxine (MAT: 568.4), taxine B (584.2), monohydroxydiacetyltaxine (MHDAT: 626.4), triacetyltaxine (TAT: 652.4), monohydroxytriacetyltaxine (MHTAT: 668.4). In both fatalities, paclitaxel, 10-DAT and cephalomannine were not identified in urine, cardiac and femoral blood but all taxoids and 3,5-DMP were present in stomach content and excreted into the bile. In urine, highest 3,5-DMP concentration was 7500 μg/L and 23,000 μg/L after enzymatic hydrolysis, respectively. In intentional and accidental poisonings, when electrocardiogram (ECG) examinations revealed ventricular tachycardia and/or prolonged QRS intervals, taxines were identified in plasma/serum, even after the ingestion of a few number of yew leaves, when 3,5-dimethoxyphenol was not even found. According to the data from one near-fatal intentional poisoning, elimination half-life of MAT, TAXIN B, MHDAT and MHTAT in serum was calculated with 11-13 h and taxines were detected up to t=+122 h post-ingestion of approximately two handfuls of yew leaves.

  • Fatal poisoning with Taxus baccata. Quantification of Paclitaxel (taxol A), 10-Deacetyltaxol, Baccatin III, 10-Deacetylbaccatin III, Cephalomannine (taxol B), and 3,5-Dimethoxyphenol in Body Fluids by Liquid Chromatography–Tandem Mass Spectrometry
    Journal of Analytical Toxicology, 2012
    Co-Authors: Thomas Grobosch, Bernd Schwarze, Daniel Stoecklein, Torsten Binscheck
    Abstract:

    This method development was to confirm the fatal ingestion of toxic yew plant material in postmortem samples (stomach content, urine, femoral blood, cardiac blood, bile, and brain tissue) collected from a 22-year-old man who committed suicide by ingesting yew leaves. The analytical method was based on a liquid-liquid extraction under alkaline conditions followed by LC-MS-MS analysis. Chromatographic separation was achieved by HPLC on a Kinetex C18 2.6u (100 × 3 mm) coupled to a QTRAP 5500 system. The method allows the simultaneous identification and quantification of the yew alkaloids taxoids paclitaxel (taxol A), 10-deacetyltaxol, baccatin III, 10-Deacetylbaccatin III, cephalomannine (taxol B), and 3,5-dimethoxyphenol; the alkaloidal diterpenoids monoacetyltaxine, taxine B, monohydroxydiacetyltaxine, triacetyltaxine, and monohydroxytriacetyltaxine were also identified. The initial hypothesis of yew tree (Taxus baccata) poisoning was confirmed. The quantitative evaluation revealed taxoid concentrations ranging from 4.5 to 132 µg/L (stomach content), 1 to 200 µg/L (urine), Language: en

Kevin D. Walker - One of the best experts on this subject based on the ideXlab platform.

  • Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N‑Debenzoyl‑N‑(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase
    2017
    Co-Authors: Chelsea K. Thornburg, Tyler Walter, Kevin D. Walker
    Abstract:

    In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-Deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis–Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)­paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis

  • The taxol pathway 10-O-acetyltransferase shows regioselective promiscuity with the oxetane hydroxyl of 4-deacetyltaxanes.
    Journal of the American Chemical Society, 2008
    Co-Authors: Mark E. Ondari, Kevin D. Walker
    Abstract:

    The 10-Deacetylbaccatin III:10β-O-acetyltransferase isolated from Taxus cuspidata regiospecifically transfers short-chain alkanoyl groups from their corresponding CoA thioesters to the C10 hydroxyl of 10-Deacetylbaccatin III. This 10-O-acetyltransferase along with five other Taxus acyltransferases on the paclitaxel (Taxol) biosynthetic pathway and one additional Taxus-derived acyltransferases of unknown function were screened for 4-O-acetyltransferase activity against 4-deacetylbaccatin III, 7-acetyl-, 13-acetyl-, and 7,13-diacetyl-4-deacetylbaccatin III. These 4-deacyl derivatives were semisynthesized from the natural product baccatin III via silyl protecting group manipulation, regioselective reductive ester cleavage with sodium bis(2-methoxyethoxy)aluminum hydride, and regioselective acetylation with acetic anhydride. Assays with the 4-deacetylated diterpene substrates and acetyl CoA revealed the taxane 10β-O-acetyltransferase was able to catalyze the 4-O-acetylation of 4-deacetylbaccatin III to baccat...

  • Profiling a Taxol Pathway 10β-Acetyltransferase: Assessment of the Specificity and the Production of Baccatin III by In Vivo Acetylation in E. coli
    Chemistry & Biology, 2006
    Co-Authors: Catherine Loncaric, Erin Merriweather, Kevin D. Walker
    Abstract:

    Summary The 10β-acetyltransferase on the biosynthetic pathway of the antineoplastic drug Taxol catalyzes the regiospecific transfer of the acetyl group of acetyl-coenzyme A (CoA) to 10-Deacetylbaccatin III. We demonstrate that in addition to acetyl group transfer, the overexpressed enzyme also catalyzes the exchange of propionyl and n -butyryl from the corresponding CoA thioester to the hydroxyl group at C10 of the cosubstrate. Also, in vivo studies revealed that E . coli , producing endogenous acetyl-CoA and overexpressing the recombinant acetyltransferase, can convert exogenously supplied 10-Deacetylbaccatin III to baccatin III. Potentially, this heterologous in vivo production method in bacteria could be optimized to couple various unnatural acyl-CoA analogs to myriad amino and/or hydroxyl acceptors by acyltransferase catalysis; conceivably, this process could facilitate the preparation of second-generation Taxols.

  • molecular cloning of a 10 deacetylbaccatin iii 10 o acetyl transferase cdna from taxus and functional expression in escherichia coli
    Proceedings of the National Academy of Sciences of the United States of America, 2000
    Co-Authors: Kevin D. Walker
    Abstract:

    The cDNA clone for a 10-Deacetylbaccatin III-10- O -acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata . By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus . This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which the full-length 10-Deacetylbaccatin III-10- O -transacetylase sequence was obtained. Expression of the ORF from pCWori + in Escherichia coli JM109 afforded a functional enzyme, as determined by 1 H-NMR and MS verification of the product baccatin III derived from 10-Deacetylbaccatin III and acetyl CoA. The full-length cDNA has an ORF of 1,320 bp corresponding to a deduced protein of 440 residues with a calculated molecular weight of 49,052, consistent with the size of the operationally soluble, monomeric, native acetyl transferase. The recombinant acetyl transferase has a pH optimum of 7.5, has K m values of 10 μM and 8 μM for 10-Deacetylbaccatin III and acetyl CoA, respectively, and is apparently regiospecific toward the 10-hydroxyl group of the taxane ring. Amino acid sequence comparison of 10-Deacetylbaccatin III-10- O -acetyl transferase with taxadienol-5- O -acetyl transferase and with other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (80% and 64–67%, respectively).

  • partial purification and characterization of acetyl coenzyme a taxa 4 20 11 12 dien 5alpha ol o acetyl transferase that catalyzes the first acylation step of taxol biosynthesis
    Archives of Biochemistry and Biophysics, 1999
    Co-Authors: Kevin D. Walker, Raymond E B Ketchum, Mehri Hezari, David Gatfield, Marta Goleniowski, Ann Barthol, Rodney Croteau
    Abstract:

    The acetylation of taxa-4(20),11(12)-dien-5alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with Km values of 4.2 and 5.5 microM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-Deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined.

Chelliah Jayabaskaran - One of the best experts on this subject based on the ideXlab platform.

  • evaluation of spore inoculum and confirmation of pathway genetic blueprint of t13αh and dbat from a taxol producing endophytic fungus
    Scientific Reports, 2020
    Co-Authors: Balabhadrapatruni V S K Chakravarthi, Satpal Singh, Subban Kamalraj, Vijai Kumar Gupta, Chelliah Jayabaskaran
    Abstract:

    Taxol (paclitaxel), a plant-derived anticancer drug, has been among the most successful anticancer drugs of natural origin. Endophytic fungi have been proposed as a prominent alternative source for Taxol and its intermediate Baccatin III, however the very low yields remain a hinderance to their commercial utilization. Significant research efforts towards this end are underway globally. Here, we report the results on our earlier reported Taxol-producing endophytic fungus, Fusarium solani from the standpoint of spores as seed inoculum and media selection for enhanced Taxol and baccatin III yields. Spores produced on M1D medium with 94.76% viability were used for further media optimization for Taxol and Baccatin III production in five different liquid media under static and shaker condition at different cultivation days. Taxol and Baccatin III when quantified through competitive inhibition enzyme immunoassay (CIEIA), showed maximum production at 136.3 µg L-1 and 128.3 µg L-1, respectively in the modified flask basal broth (MFBB) under shaking condition. Further, two important genes of this pathway, namely taxane 13α-hydroxylase (T13αH) and 10-Deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) have been identified in this fungus. These findings are hoped to assist in further manipulation and metabolic engineering of the parent F. solani strain towards the enhanced production of Taxol and baccatin III.

  • Biochemical insights into the recombinant 10-Deacetylbaccatin III-10-β-O-acetyltransferase enzyme from the Taxol-producing endophytic fungus Lasiodiplodia theobromae
    Fems Microbiology Letters, 2019
    Co-Authors: Kamalraj Subban, Chelliah Jayabaskaran
    Abstract:

    10-Deacetylbaccatin III-10-beta-O-acetyltransferase (DBAT) is a key rate-limiting enzyme of the Taxol biosynthetic pathway, which is uncharacterized in Taxol-producing endophytic fungi. Here, an open reading frame of DBAT was cloned from the Taxol-producing endophytic fungus Lasiodiplodia theobromae (LtDBAT). The LtDBAT enzyme was heterologously expressed and purified by the affinity and gel filtration chromatography methods. The molecular weight of the purified protein was 49 kDa and its identity was confirmed by western blot. The purified LtDBAT enzyme was capable of catalyzing 10-Deacetylbaccatin III into baccatin III, as shown by liquid chromatography-mass spectroscopy. The mass spectra of baccatin III were identical to the authentic baccatin III. The LtDBAT enzyme was characterized and the kinetic parameters of catalysis were determined. In addition, localization of LtDBAT was performed by using confocal microscopy and the result showed that the enzyme was localized in lipid droplets. Together, this study provides biochemical insights into the fungal recombinant DBAT enzyme that is involved in the Taxol biosynthetic pathway. In the near future, engineering of the LtDBAT enzyme and the Taxol biosynthetic pathway in endophytic fungi could be an eco-friendly and economically feasible alternative source for production of Taxol and its precursors.