Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.3.17 (taxadiene synthase)
58 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (</=5%) side-product subsequent to its unexpected crystallization. Accordingly, we now report the serendipitous crystal structure determination of unliganded lactaldehyde dehydrogenase from E. coli determined by the technique of multiple isomorphous replacement using anomalous scattering at 2.2 A resolution. Additionally, we report the crystal structure of the ternary enzyme complex with products lactate and NADH at 2.1 A resolution, and the crystal structure of the enzyme complex with NADPH at 2.7 A resolution. The structure of the ternary complex reveals that the nicotinamide ring of the cofactor is disordered between two conformations: one with the ring positioned in the active site in the so-called hydrolysis conformation, and another with the ring extended out of the active site into the solvent region, designated the out conformation. This represents the first crystal structure of an aldehyde dehydrogenase-product complex. The active site pocket in which lactate binds is more constricted than that of medium-chain dehydrogenases such as the YdcW gene product of E. coli. The structure of the binary complex with NADPH reveals the first view of the structural basis of specificity for NADH: the negatively charged carboxylate group of E179 destabilizes the binding of the 2'-phosphate group of NADPH sterically and electrostatically, thereby accounting for the lack of enzyme activity with this cofactor.
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PMID:Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity. 1717 28

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.
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PMID:Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei. 1792 79

A cladistic analysis of the medicinal plant Taxus, using the sequences of one chloroplast (trnS-trnQ spacer) and three nuclear taxadiene synthase (TS), 10-deacetylbaccatin III-10beta-O-acetyltransferase (DBAT), and 18S rDNA) molecular markers, was carried out by distance, parsimony, likelihood, and Bayesian methods. Three of the four New World species (T. brevifolia, T. floridana and T. globosa) form a well-supported clade, whereas T. canadensis initially branches-appearing distantly related to both Old World taxa and New World species. In Asia, Taxus chinensis, T. mairei, T. sumatrana and T. wallichiana cluster together and are sister to a clade containing T. baccata and T. contorta. Taxus yunnanensis is more closely related to T. wallichiana than to four other Taxus species in our study from China; T. contorta is closer to the Euro-Mediterranean T. baccata than to the Asian species. This study provides a genetic method for authentication of economically important Taxus species and proposes a robust phylogenetic hypothesis for the genus. Using trnS-trnQ spacer sequences, we were able to distinguish T. mairei from all other species of Taxus.
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PMID:Phylogenetic relationships of the genus Taxus inferred from chloroplast intergenic spacer and nuclear coding DNA. 1823 84

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10 beta-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.
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PMID:Molecular evolution of paclitaxel biosynthetic genes TS and DBAT of Taxus species. 1832 45

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7+/-0.85mg/l as well as significant amounts of geranylgeraniol (33.1+/-5.6mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.
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PMID:Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production. 1848 76

Three different strains of the endophytic fungus Phomopsis were isolated from the healthy leaves of Taxus cuspidata (Japanese yew), Ginkgo biloba (ginkgo or maidenhair tree) and Larix leptolepis (Japanese larch) and screened for the production of taxol on a modified liquid medium for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 418 microg/litre in the strain BKH 27. The yield was increased to 8360-fold that found for the fungus Taxomyces andreanae reported previously [Stierle, Strobel and Stierle (1993) Science 260, 214-216]. The fungal taxol extracted also showed a strong cytotoxicity towards the human cancer cells in an apoptosis assay. All the three isolates showed positive sign towards PCR for the conserved sequence of the taxadiene synthase gene. The results suggest that Phomopsis could be an excellent alternative source for taxol and may serve as a potential genetic-engineered species for the enhanced production of taxol.
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PMID:Screening of species of the endophytic fungus Phomopsis for the production of the anticancer drug taxol. 1906 52

To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200mgL(-1)). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200mgL(-1) exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.
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PMID:Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures. 1910 60

More than 50 kinds of endophytic fungi associated with Taxus chinensis, were isolated and examined as potential source of the imposing anticancer drug taxol. Of these, 4 isolates show ability to produce taxane when measured with the competitive inhibition enzyme immunoassay method. The most promising clone, DA10, identified as Mucor rouxianus sp., is the first rouxianus reported as taxol-production fungus. The presence of taxol and its important precursors, such as 10-diacetyl baccatinIII (10-DAB) and baccatinIII, in the culture of this fungus was confirmed by reactivity with taxane-specific monoclonal antibody, comparative chromatographic and mass spectrometric behavior, cytotoxity to liver carcinoma 7402, and molecular cloning of kernel fragment of taxadiene synthase gene.
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PMID:A new endophytic taxane-production fungus from Taxus chinensis. 1923 15

The 1990s brought an abundance of reports on paclitaxel-producing endophytes, initially heralded as a discovery having tremendous implications for cancer therapy. As the vision of large-scale fermentation tanks producing vast quantities of relatively inexpensive paclitaxel and novel taxanes has faded and has been replaced by controversial silence, we carried out an in-depth investigation of Taxomyces andreanae - the very first presumed endophytic synthesizer of the diterpenoid. On one hand, metabolic profiling by means of chromatographic, spectroscopic and immunoenzymatic techniques predominant in literature was taken up. On the other, the experimental procedure was brought to an alternative, previously unattempted level aiming at revealing the genetic background of paclitaxel biosynthesis in the endophyte. The profound PCR-based screening for taxadiene synthase (TXS) - a gene unique to the formation of the primary taxane-skeleton, as well as phenylpropanoyl transferase (BAPT) encoding for the catalyst of the final acylation of the core structure rendering the ultimate efficacy of the drug, confirmed the molecular blueprint for paclitaxel biosynthesis to be an inherent genetic trait of the endophyte. However, as the thorough metabolic analysis of Taxomyces andreanae commercial isolate brought no confirmation of endophytic paclitaxel production even after considerable up-scaling endeavors, we postulate that proclaiming the strain "a fungus factory for Taxol" might have been premature.
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PMID:Taxomyces andreanae: a presumed paclitaxel producer demystified? 1980 69

Availability of isotope-labelled metabolites often proves to be an essential prerequisite for the successful identification of a biosynthetic pathway. Also for the metabolic engineering isotope-labelled, either radioactive or non-radioactive, biosynthetic intermediates represent valuable tools for the assessment of metabolic flux, identification of unexpected biosynthetic side reactions, or for the confirmation of the functionality of the engineered reaction step. Most often the required compounds are neither available commercially nor prepared by chemical means within an acceptable time span and effort. Biocatalytic synthesis of early pathway intermediates may offer an attractive solution for this problem. For the metabolic engineering of taxol biosynthesis in a heterologous host, like yeast, isotope-labelled taxanes represent useful tools for the establishment and functional assessment of the introduced biosynthetic steps. Using Taxus chinensis taxadiene synthase expressed in the heterologous organism Pichia pastoris, we describe a method for the biocatalytic synthesis of tritium ((3)H)-labelled taxa-4(5),11(12)-diene, which represents the pathway committing biosynthetic precursor for all taxoid diterpenoids.
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PMID:Biocatalytic synthesis of tritium ((3)H)-labelled taxa-4(5),11(12)-diene, the pathway committing precursor of the taxoid diterpenoids. 2055 51


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