The Experts below are selected from a list of 27 Experts worldwide ranked by ideXlab platform
R. Van Der Heijden - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, M. M. L. Ibañez, R. Van Der HeijdenAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to Cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
Hasnain G. - One of the best experts on this subject based on the ideXlab platform.
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The ORCA-ome as a key to understanding alkaloid biosynthesis in Catharanthus roseus
2010Co-Authors: Hasnain G.Abstract:Catharanthus roseus produces an important class of secondary metabolites known as terpenoid indole alkaloids (TIA). The dimeric TIA vincristine and vinblastine are effective anticancer drugs. Two transcription factors called ORCA2 and ORCA3 have been reported to regulate the MeJA-responsive expression of several biosynthesis genes. We report here comprehensive transcript profiling analysis of Catharanthus cell lines overexpressing ORCA2 or ORCA3 in an inducible manner. By using cDNA-amplified fragment-length polymorphism technology the quantitative accumulation patterns of 11,277 transcript tags were determined and analyzed. Thirty six transcripts were upregulated in response to overexpression of either by both ORCA2 or ORCA3 while 22 tags and 16 tags were upregulated specifically in ORCA2 overexpressing lines and ORCA3 over expression lines, respectively. Accumulation of downstream TIAs was also differentially affected by ORCA2 and ORCA3 overexpression. TIA accumulation patterns allowed us to predict in which set of cDNA-AFLP tags genes encoding the corresponding enzymes should be present. Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels, indicating that the gene encoding Cathenamine Reductase (CR) must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to CR. Recombinant CR-75 protein produced in E. coli was able convert Cathenamine to ajmalicine using NADPH as a cofactor, thereby identifying CR-75 as Cathenamine Reductase. We also report the isolation and characterization of alcohol dehydrogenase genes from C. roseus which may function as 10-hydroxygeraniol oxidoreductases (10HGO). Two ORCA regulated genes were also isolated and characterized from C. roseus cell suspension culture which may have roles in TIA biosynthesi
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The ORCA-ome as a key to understanding alkaloid biosynthesis in Catharanthus roseus
2010Co-Authors: Hasnain G.Abstract:Catharanthus roseus produces an important class of secondary metabolites known as terpenoid indole alkaloids (TIA). The dimeric TIA vincristine and vinblastine are effective anticancer drugs. Two transcription factors called ORCA2 and ORCA3 have been reported to regulate the MeJA-responsive expression of several biosynthesis genes. We report here comprehensive transcript profiling analysis of Catharanthus cell lines overexpressing ORCA2 or ORCA3 in an inducible manner. By using cDNA-amplified fragment-length polymorphism technology the quantitative accumulation patterns of 11,277 transcript tags were determined and analyzed. Thirty six transcripts were upregulated in response to overexpression of either by both ORCA2 or ORCA3 while 22 tags and 16 tags were upregulated specifically in ORCA2 overexpressing lines and ORCA3 over expression lines, respectively. Accumulation of downstream TIAs was also differentially affected by ORCA2 and ORCA3 overexpression. TIA accumulation patterns allowed us to predict in which set of cDNA-AFLP tags genes encoding the corresponding enzymes should be present. Metabolite analysis showed that overexpression of either ORCA2 or ORCA3 resulted in increased ajmalicine levels, indicating that the gene encoding Cathenamine Reductase (CR) must be regulated by both ORCAs. Since the CR-75 tag gave a TBLASTX hit to aldo/keto oxidoreductase enzymes we investigated the possibility that CR-75 corresponds to CR. Recombinant CR-75 protein produced in E. coli was able convert Cathenamine to ajmalicine using NADPH as a cofactor, thereby identifying CR-75 as Cathenamine Reductase. We also report the isolation and characterization of alcohol dehydrogenase genes from C. roseus which may function as 10-hydroxygeraniol oxidoreductases (10HGO). Two ORCA regulated genes were also isolated and characterized from C. roseus cell suspension culture which may have roles in TIA biosynthesisPartially supported by NWFP Agricultural University Peshawar PakistanUBL - phd migration 201
Arjan Geerlings - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, M. M. L. Ibañez, R. Van Der HeijdenAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to Cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
Johan Memelink - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, M. M. L. Ibañez, R. Van Der HeijdenAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to Cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.
M. M. L. Ibañez - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Analysis of Strictosidine β-d-Glucosidase, an Enzyme in Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus
The Journal of biological chemistry, 2000Co-Authors: Arjan Geerlings, Johan Memelink, M. M. L. Ibañez, R. Van Der HeijdenAbstract:Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to Cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.