The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Margaret Essenberg - One of the best experts on this subject based on the ideXlab platform.
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inhibitor and substrate activities of sesquiterpene olefins toward δ Cadinene 8 hydroxylase a cytochrome p450 monooxygenase cyp706b1
Phytochemistry, 2010Co-Authors: Yanhong Wang, Margaret EssenbergAbstract:Several lines of evidence indicate that (+)-δ-Cadinene-8-hydroxylase (CYP706B1) plays an important role in biosynthesis of gossypol in Gossypium arboreum L. (Luo et al., 2001; Wang et al., 2003). The catalytically active enzyme has been expressed in yeast microsomes. Some microsomal preparations conjugated the hydroxylated (+)-δ-Cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-β-d-glucoside (OG), a non-ionic detergent, (+)-δ-Cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-δ-Cadinene. OG had little effect on K(m) and slightly stimulated apparent V(max). Enzymic activity was more than 10-fold more sensitive to inhibition by the N-substituted imidazole clotrimazole than to miconazole. Sesquiterpene olefins (-)-δ-Cadinene, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and a mixture of (-)- and (+)-α-copaene were inhibitory to hydroxylation of (+)-δ-Cadinene. In addition, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and, to a smaller extent, (-)-δ-Cadinene served as alternative substrates for (+)-δ-Cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, α-humulene and α-copaene are found in lysigenous glands of cotton (Elzen et al., 1985), which are also the site of gossypol accumulation (Bell et al., 1978; Mace et al., 1976) and the probable site of its biosynthesis.
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8 hydroxy δ Cadinene is a precursor to hemigossypol in gossypium hirsutum
Phytochemistry, 2003Co-Authors: Yanhong Wang, Guadalupe Davilahuerta, Margaret EssenbergAbstract:Abstract [3H](+)-δ-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-3H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [3H](+)-8-hydroxy-δ-Cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [3H](+)-8-hydroxy-δ-Cadinene had been converted to hemigossypol.
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molecular cloning and functional identification of δ Cadinene 8 hydroxylase a cytochrome p450 mono oxygenase cyp706b1 of cotton sesquiterpene biosynthesis
Plant Journal, 2001Co-Authors: Ping Luo, Margaret Essenberg, Yanhong Wang, Guodong Wang, Xiaoya ChenAbstract:In cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)-delta-Cadinene, forming 8-hydroxy-(+)-delta-Cadinene. This P450 mono-oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)-delta-Cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.
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δ Cadinene is a product of sesquiterpene cyclase activity in cotton
Phytochemistry, 1995Co-Authors: Gordon D Davis, Margaret EssenbergAbstract:Abstract Glandless cotton cotyledons stimulated to produce sesquiterpenoid phytoalexins by inoculation with Xanthomonas campestris pv. malvacearum, or by injection of oligogalacturonide elicitors, generated a hydrocarbon that was absent in mock-inoculated or non-inoculated cotyledons. Enzyme preparations from the same cotton cotyledons catalysed cell-free reactions which converted (E, E)-[1-3H]farnesyl pyrophosphate into a predominant tritium-labelled hydrocarbon product. Large-scale cell-free reactions catalysed by enzyme preparations from cotton cotyledons previously inoculated with Xanthomonas campestris pv. malvacearum converted nonradioactive (E, E)-farnesyl pyrophosphate into the hydrocarbon product, which was identified as (+)-δ-Cadinene by chiral GC-mass spectrometry. In planta incorporation of tritium into the sesquiterpenoid phytoalexins 2,7-dihydroxycadalene, lacinilene C, lacinilene C7-methyl ether and structurally related terpenoids occurred following injection of [3H] (+)-δ-Cadinene into previously inoculated glandless cotton cotyledons. The accumulation of (+)-δ-Cadinene in bacteria-inoculated or elicitor-treated cotton cotyledons and the results of the incorporation experiment suggest that (+)-δ-Cadinene is an early enzymatic intermediate in the biosynthesis of the sesquiterpenoid phytoalexins by upland cotton.
Yasuo Yoshikuni - One of the best experts on this subject based on the ideXlab platform.
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-Cadinene synthase suggested that the G helix plays a very important role in (+)-δ-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-Cadinene synthase suggested that the G helix plays a very important role in (+)-delta-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
Jay D Keasling - One of the best experts on this subject based on the ideXlab platform.
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-Cadinene synthase suggested that the G helix plays a very important role in (+)-δ-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-Cadinene synthase suggested that the G helix plays a very important role in (+)-delta-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
Vincent J J Martin - One of the best experts on this subject based on the ideXlab platform.
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-Cadinene synthase suggested that the G helix plays a very important role in (+)-δ-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-Cadinene synthase suggested that the G helix plays a very important role in (+)-delta-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
Thomas E Ferrin - One of the best experts on this subject based on the ideXlab platform.
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-Cadinene synthase suggested that the G helix plays a very important role in (+)-δ-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
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engineering cotton δ Cadinene synthase to an altered function germacrene d 4 ol synthase
Chemistry & Biology, 2006Co-Authors: Thomas E Ferrin, Yasuo Yoshikuni, Vincent J J Martin, Jay D KeaslingAbstract:The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-Cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-Cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-Cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-Cadinene synthase suggested that the G helix plays a very important role in (+)-delta-Cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).
