Tropane Alkaloid Production in Hyoscyamus Root Cultures

Hashimoto, Takashi; Yukimune, Yukihito; Yamada, Yasuyuki

doi:10.1016/s0176-1617(86)80178-x

Cited by 130 publications

(38 citation statements)

References 10 publications

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“…Putrescine N-methyltransferase (14) and hyoscyamine 6#-hydroxylase (11), other enzymes involved in the biosynthesis of hyoscyamine and scopolamine, are also highly active in the young root tissues. Together with the classical grafting experiments between alkaloid-producing and nonproducing plants (21), and alkaloid formation in organ cultures (13), these results suggest that hyoscyamine and scopolamine are mostly synthesized in the young root cells and translocated to the aerial parts of the plant. Recent immunohistochemical localization of hyoscyamine 6,B-hydroxylase to the pericycle cells of the root (10) further supports this conclusion.…”

Section: Discussionsupporting

confidence: 55%

“…Cultured roots of Hyoscyamus niger L. have been maintained in our laboratory since 1984 as described elsewhere (13). Several plant species (Physochlaina orientalis M.B.…”

Section: Plant Materialsmentioning

confidence: 99%

“…The reaction products were extracted as described above and analyzed by GC-MS as described elsewhere (13). The electron impact ionization mode was set at 70 eV.…”

Section: Determination Of Stereospecific Hydrogen Transfermentioning

confidence: 99%

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Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger

Hashimoto¹,

Nakajima²,

Ongena³

et al. 1992

Plant Physiol.

118

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Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids. Two stereospecifically distinct NADPH-dependent oxidoreductases, TR-I and TR-11, which, respectively, reduce tropinone to 3a-hydroxytropane (tropine) and 3j@-hydroxytropane (4-tropine), were detected mainly in the root of tropane alkaloid-producing plants but not in nonproducing cultured root. Both reductases were purified to near homogeneity from cultured root of Hyoscyamus niger and characterized. The TR-1 reaction was reversible, whereas the TR-11 reaction was essentially irreversible, reduction of the ketone being highly favored over oxidation of the alcohol 4-tropine. Marked differences were found between the two reductases in their affinities for tropinone substrate and in the effects of amino acid modification reagents. Some differences in substrate specificity were apparent. For example, N-propyl-4-piperidone was reduced by but not by TR-1. Conversely, 3-quinuclidinone and 8-thiabicyclo[3,2, 1]octane-3-one were accepted as substrates by TR-I but hardly at all by TR-11. Both enzymes were shown to be class B oxidoreductases, which transfer the pro-S hydrogen of NAD(P)H to their substrates. Possible roles of these tropinone reductases in alkaloid biosynthesis are discussed.Tropinone is an alkamine intermediate at the branch point of biosynthetic pathways leading to various tropane alkaloids mainly found in Solanaceae (Fig. 1). The 3-keto group of tropinone can be reduced to hydroxyls having two different configurations: tropine has a hydroxy group on the opposite side of the tropane nitrogen atom (a-configuration), whereas 0-tropine has the group on the same side (fl-configuration). (TR-II) was later found in cultured roots of Hyoscyamus niger (5). Subsequent studies by us (23) and others (4) indicated that the two stereospecifically distinct reductase activities can coexist in a given alkaloid-producing species. How the metabolic fates of tropinone are controlled by these reductases is an interesting question, particularly in view of our observation that the {-tropine-forming activity seemed to be predominant in cultured H. niger roots that accumulated mostly tropine-derived alkaloids (5). Here we report distribution of the two reductases in various plant species, their organspecific expression, and detailed biochemical characterization of the enzymes highly purified from cultured roots of H. niger. MATERIALS AND METHODS Plant MaterialsCultured roots of Hyoscyamus niger L. have been maintained in our laboratory since 1984 as described elsewhere (13). Several plant species (Physochlaina orientalis M.B. Don, Atropa acuminata Royle ex Lindl, Physalis alkekengi L., and Calystegia sepium R. Br) were transformed with Agrobacterium rhizogenes (strain 15834) by Dr. P. Bachmann at our laboratory. A hairy root clone (D15/5) of Datura stramonium was obtained from Dr. R. Robins, Institute of Food Research, Norwich, UK. These roots were cultured in auxin-free B5 medium (7) supplemented with ...

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Section: Discussionsupporting

confidence: 55%

“…Cultured roots of Hyoscyamus niger L. have been maintained in our laboratory since 1984 as described elsewhere (13). Several plant species (Physochlaina orientalis M.B.…”

Section: Plant Materialsmentioning

confidence: 99%

See 1 more Smart Citation

Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger

Hashimoto¹,

Nakajima²,

Ongena³

et al. 1992

Plant Physiol.

118

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“…Tropane alkaloids were extracted from plant tissues and then purified and quantified by gas-liquid chromatography, as described (24). Alkaloids were identified by comparing their mass spectra with those of authentic samples (25).…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

Yun

Hashimoto

Yamada

1992

Proc. Natl. Acad. Sci. U.S.A.

275

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The tropane alkaloid scopolamine is a medicinally important anticholinergic drug present in several solanaceous plants. Hyoscyamine 6l-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. We introduced the hydroxylase gene from Hyoscyamus niger under the control of the cauliflower mosaic virus 35S promoter into hyoscyaminerich Atropa belladonna by the use of an Agrobacterummediated transformation system. A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase. In the primary transformant and its selfed progeny that inherited the transgene, the alkloid contents of the leaf and stem were almost exclusively scopolamine. Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.

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“…6) Our results show that there is no statistical correlation (r = 0.208 for untransformed and r -0.201 for transformed root cultures) between scopolamine content and growth, as has been reported for H. niger root cultures. 6) …”

Section: Relationship Between Scopolamine Content and Growthmentioning

confidence: 99%

Tropane Alkaloid Production in Root Cultures ofDuboisia myoporoidesObtained by Repeated Selection

Yukimune¹,

Hara²,

Yamada

1994

Bioscience, Biotechnology, and Biochemistry

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Tropane Alkaloid Production in Hyoscyamus Root Cultures

Cited by 130 publications

References 10 publications

Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger

Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

Tropane Alkaloid Production in Root Cultures ofDuboisia myoporoidesObtained by Repeated Selection

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