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Rodney Croteau - One of the best experts on this subject based on the ideXlab platform.
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monoterpene metabolism cloning expression and characterization of isopiperitenol Carveol dehydrogenase of peppermint and spearmint
Plant Physiology, 2005Co-Authors: Kerry L Ringer, Edward M Davis, Rodney CroteauAbstract:The essential oils of peppermint ( Mentha x piperita ) and spearmint ( Mentha spicata ) are distinguished by the oxygenation position on the p -menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (−)-trans-isopiperitenol to (−)-isopiperitenone in peppermint and (−)-trans-Carveol to (−)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding ( − )-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (−)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (−)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5′-truncated cDNA encoding the spearmint homolog, (−)-trans-Carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5′-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (−)-trans-isopiperitenol and (−)-trans-Carveol. These isopiperitenol/Carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (−)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.
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monoterpene biosynthesis pathway construction in escherichia coli
Phytochemistry, 2003Co-Authors: Ora A Carter, Reuben J Peters, Rodney CroteauAbstract:Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-Carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and Carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions.
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cytochrome p 450 dependent limonene 6 hydroxylation in fruits of caraway carum carvi
Phytochemistry, 1999Co-Authors: Harro J. Bouwmeester, Maurice C.j.m Konings, Jonathan Gershenzon, Frank Karp, Rodney CroteauAbstract:Abstract Microsomal preparations from fruits of annual and biennial forms of caraway (Carum carvi L.) catalyze the C-6 hydroxylation of (+)-limonene to (+)-trans-Carveol, the key intermediate in the biosynthesis of carvone. The enzyme activities from both caraway forms had similar properties (pH optimum, Km, cofactor requirements, etc). Both activities were dependent on NADPH and O2, and were inhibited by N-substituted imidazoles, metyrapone, cytochrome c and CO, and thus meet many of the established criteria for cytochrome P-450-dependent mixed function oxygenases.
O Murat - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of Human α7 Nicotinic Acetylcholine Receptors by Cyclic Monoterpene Carveol
Chapman University Digital Commons, 2016Co-Authors: Lozon Yusra, Sultan Ahmed, Lansdell, Stuart J., Prytkova Tatiana, Sadek Bassem, Yang, Keun-hang Susan, Howarth, Frank Christopher, Millar, Neil S., O MuratAbstract:Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, Carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order Carveol\u3ethymoquinone\u3ecarvacrol\u3ementhone\u3ethymol\u3elimonene\u3eeugenole\u3epulegone≥carvone≥vanilin. Among the monoterpenes, Carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as Carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies
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Carveol inhibits the function of human α7 nicotinic acetylcholine receptors
HAMDAN MEDICAL JOURNAL, 2015Co-Authors: A Alhosani, A Alali, S Nurulain, O MuratAbstract:Introduction: Carveol is a natural unsaturated, monocyclic monoterpenoid alcohol which is a constituent of spearmint essential oil. Earlier studies have shown that Carveol has analgesic and anti-inflammatory actions in various in vivo studies. Currently, mechanisms of these effects are largely unknown. Objectives: The effects of Carveol application on the functional properties of these receptors have been investigated. Materials and methods: cRNA encoding for homomeric human α 7-nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes. Ion currents mediated by the activation of nACh receptors were recorded using a two-electrode voltage clamp method. Results: Our results showed that Carveol caused a significant inhibition of nACh receptor-mediated ion currents. The effect of Carveol was reversible and gradually reached a steady-state level within 10 minutes of application time. Maximal amplitudes of currents activated by 100μM nACh were significantly inhibited by Carveol in a concentration-dependent manner with an EC50 (half maximal effective concentration) value of 7.9μM. Conclusions: Our results indicate that Carveol inhibits the function of human α 7-nACh receptors. Acknowledgements: Professor Murat Oz for supervising this project.
Murat Oz - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene Carveol
European Journal of Pharmacology, 2016Co-Authors: Yosra Lozon, Ahmed Sultan, Stuart J. Lansdell, Tatiana R. Prytkova, Bassem Sadek, Keun-hang Susan Yang, Frank Christopher Howarth, Neil S. Millar, Murat OzAbstract:Abstract Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, Carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order Carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, Carveol showed the highest potency on acetylcholine-induced responses, with IC 50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as Carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.
Manuela M R Da Fonseca - One of the best experts on this subject based on the ideXlab platform.
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adaptation of rhodococcus erythropolis dcl14 to growth on n alkanes alcohols and terpenes
Applied Microbiology and Biotechnology, 2005Co-Authors: Carla C. C. R. De Carvalho, Manuela M R Da Fonseca, Beatriz Parrenomarchante, Grit Neumann, Hermann J HeipieperAbstract:Rhodococcus erythropolis DCL14 has the ability to convert the terpene (−)-Carveol to the valuable flavour compound (−)-carvone when growing on a wide range of carbon sources. To study the effect of carbon and energy sources such as alkanes, alkanols and terpenes on the biotechnological process, the cellular adaptation at the level of fatty acid composition of the membrane phospholipids and the (−)-carvone production were examined. All tested carbon sources caused a dose-dependent increase in the degree of saturation of the fatty acids. The exception was observed with short-chain alcohols such as methanol and ethanol, to which the cells adapted with a concentration-dependent decrease in the saturation degree of the membrane phospholipids. This influence of the different carbon sources on the rigidity of the cell membrane also had an impact on the (−)-carvone productivity of the strain.
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towards the bio production of trans Carveol and carvone from limonene induction after cell growth on limonene and toluene
Tetrahedron-asymmetry, 2003Co-Authors: Carla C. C. R. De Carvalho, Manuela M R Da FonsecaAbstract:Abstract Rhodococcus opacus PW4 cells were found to produce trans - and cis -Carveol and/or carvone as result of limonene metabolism, depending on the type and concentration of the carbon source used for cell growth. In aqueous systems, cells grown on ethanol and toluene only produced trans -Carveol, whilst cells grown on limonene and on toluene with a larger head-space available produced both trans -Carveol and carvone. In biphasic systems, limonene was converted to trans - and cis -Carveol as well as to carvone, regardless of the carbon source used, although Carveol and carvone production rates were higher in toluene and limonene grown cells, respectively. A good and stable emulsion was obtained in a magnetically stirred two-phase reactor but both trans -Carveol and carvone were produced at low rates: 0.08 and 0.02 nmol/min mg prot, respectively. No cis -Carveol was formed. When (−)-Carveol was added, carvone production increased 4.7 fold to 0.12 nmol/min mg prot. Using an aerated two-phase reactor, carvone production was enhanced even with cells grown on toluene. The highest trans - and cis -Carveol and carvone production rates were attained with cells grown on limonene by continuously supplying limonene to the reactor through the air stream, carvone production reaching 0.58 nmol/min mg prot. The best trans- / cis -Carveol ratio (2.26) was observed with cells grown on toluene when limonene was supplied in the gas phase. When 50 mM limonene was added initially, carvone was produced 27.9 and 141.4 times faster than trans -Carveol with cells grown on toluene and limonene, respectively.
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maintenance of cell viability in the biotransformation of Carveol with whole cells of rhodococcus erythropolis
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Carla C. C. R. De Carvalho, Manuela M R Da FonsecaAbstract:Abstract Whole cells of Rhodococcus erythropolis DCL14 present Carveol dehydrogenase (CDH) activity, which allows them to stereoselectively carry out the oxidation of the (+)-cis and (−)-trans-Carveol to (+)- and (−)-carvone, respectively [1] . When a diastereomeric mixture of (−)-Carveol was supplied for biotransformation, the (−)-trans-Carveol was converted to (−)-carvone. When the cells grow on limonene or cyclohexanol the major activity is NAD-dependent. The relatively low water solubility of Carveol and carvone was overcome through the implementation of an organic:aqueous system. The prolonged productivity of such a system depends on cell viability, since viable cells are naturally able to regenerate the co-factor. Fluorescence microscopy was used to off-line monitor cell viability during the time course of the biotransformation. n-Dodecane was the solvent that allowed the highest retention of both cell activity and viability. The most adequate phase ratio was 1:5, at which, for ODs higher than 0.57, a stable emulsion is formed. At an OD of 0.46 only half of the solvent was emulsified. Loss of viability increased with the OD, probably due to oxygen depletion. The maximum specific production rate was obtained at an initial Carveol concentration of 125 mM. At this scale (60 ml flasks) the best aeration rate was 0.01 vvm, both with regard to viability and stability of the emulsion. Carvone was found to be toxic, causing cell death at concentrations above 50 mM.
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influence of reactor configuration on the production of carvone from Carveol by whole cells of rhodococcus erythropolis dcl14
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Carla C. C. R. De Carvalho, Manuela M R Da FonsecaAbstract:Abstract Biocatalysis with whole cells in aqueous–organic systems is extremely attractive for conversion of substrates with low water solubility, specially if co-factor regeneration is required. However, in the latter case, cells should be able to stay viable, which makes both reactor configuration and operation conditions important. Three reactor configuration types were tested: mechanically stirred direct contact reactor, silicone tube membrane reactor and air-driven column reactor. All reactors were operated with pulse additions of Carveol. The maximum trans-Carveol conversion (92%) and specific production rate (1.69 mg carvone/h mg prot) were obtained with the mechanically stirred reactor at, respectively, ambient temperature, in 50 mM, pH 7.0 phosphate buffer, and at 28 °C in mineral medium. The highest productivity (0.164 mg carvone/h ml org) was attained with the air-driven column reactor after the cells had been adapted to the presence of solvent, substrate and product.
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development of a reaction system for the selective conversion of trans Carveol to carvone with whole cells of rhodococcus erythropolis dcl14
Journal of Molecular Catalysis B-enzymatic, 2001Co-Authors: Carla Tecelao, Frederik Van Keulen, Manuela M R Da FonsecaAbstract:Abstract The present article addresses the development of a microbial reaction system for the transformation of Carveol to carvone, using whole cells of Rhodococcus erythropolis DCL14. This strain contains a NAD-dependent Carveol dehydrogenase (CDH) when grown on limonene or on cyclohexanol. When a mixture of (−)- cis and (−)- trans -Carveol is supplied, only (−)- trans -Carveol is converted. Thus, besides (−)-carvone, pure (−)- cis -Carveol can be obtained as product. Initial experiments were performed batchwise using an aqueous system. (−)- Trans -Carveol conversion rate gradually decreased during successive reutilisation batches. After the third reutilisation, activity was completely lost. Cells grown on cyclohexanol showed a slightly higher activity as compared to cells grown on (+)-limonene. A production of 4.3 μmol (−)-carvone formed per mg protein was achieved. A significant improvement with respect to initial reaction rate and productivity was obtained with aqueous–organic two-phase systems. Using a 5 to 1 buffer/ iso -octane system, a 40% increase in the initial rate and a 16-fold increase of the production was observed. A further improvement resulted from increasing the volume of solvent (1 to 1 buffer/dodecane ratio). An initial reaction rate of 26 nmol/(min∗mg protein) was observed, while production increased to 208 μmol (−)-carvone formed per mg protein. As in the single-phase system, reaction rate gradually decreased along the successive cell reutilisation batches. Addition of co-substrates for the regeneration of NAD did not prevent this decay. A simple downstream process was developed for the recovery of carvone and cis -Carveol.
Yosra Lozon - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene Carveol
European Journal of Pharmacology, 2016Co-Authors: Yosra Lozon, Ahmed Sultan, Stuart J. Lansdell, Tatiana R. Prytkova, Bassem Sadek, Keun-hang Susan Yang, Frank Christopher Howarth, Neil S. Millar, Murat OzAbstract:Abstract Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, Carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order Carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, Carveol showed the highest potency on acetylcholine-induced responses, with IC 50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as Carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.
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Carveol and related monoterpenes inhibit the function of the human α7 nicotinic acetylcholine receptor
2015Co-Authors: Adnan Lozon, Yosra LozonAbstract:Plants and phytochemicals have been used for centuries for therapeutic purposes. Essential oils from these plants are complex mixtures and may possess a large spectrum of biological activities many of them of clinical interest. Among the active constituents of essential oils, monoterpenes, demonstrated valuable antioxidant, antiviral, antimicrobial, anticancer, analgesic and anti-inflammatory effects. In recent years, actions of monoterpenes on the function of ion channels have been investigated. In the present study, effects of different monoterpenes including carvacrol, Carveol, d-carvone, eugenol, (+)-pulegone, thymol, thymoquinone, menthome and limonene, on the function of the cloned α7 subunit of human nicotinic acetylcholine (nACh) receptor expressed in Xenopus oocytes were investigated by using the two-electrode voltage-clamp technique. All monoterpenes caused a variable extent of reversible inhibition of ACh (100 μM)-induced currents except vanillin and d-carvone. Carveol showed maximum potency of inhibition with an IC50 value of 8.3 μM. The effect of Carveol was further investigated and found to be independent of the membrane potential. Carveol (10 μM) did not affect the activity of endogenous Ca 2+ -dependent Cl channels since the extent of inhibition by Carveol was unaltered by the intracellularly injected Ca 2+ chelator BAPTA and perfusion with Ca 2+ -free bathing solution containing 2 mM Ba 2+ . The effect of Carveol was associated with decreased potency of the ACh, and the inhibition was fully reversed by increasing ACh concentrations, suggesting that this drug acts in a competitive manner. In conclusion, these results demonstrate for the first time that monoterpenes inhibit directly the function of human α7-nACh receptors expressed in Xenopus oocytes. It appears that the extent of inhibition by monoterpenes differs significantly depending on their chemical structures.