Partial Purification and Properties of Tropine Dehydrogenase from Root Cultures of<i>Datura stramonium</i>

Koelen, Karl J.; Gross, Georg G.

doi:10.1055/s-2007-971454

Cited by 33 publications

(13 citation statements)

References 15 publications

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“…Two stereospecific NADPH-dependent reductases, tropinone reductase TR-I and TR-II, constitute a branching point in the biosynthesis of tropane alkaloids: TR-I catalyses the stereospecific reduction of tropinone to tropine, whereas TR-II reduces tropinone to pseudotropine (Koelen and Gross 1982;Drager et al 1988). Hashimoto et al (1992) previously characterized TRs that had been purified from cultured roots of H. niger and showed that the two TRs had both common and different biochemical and kinetic properties.…”

Section: Gene Cloningmentioning

confidence: 99%

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Zhang

Kai

Lü

et al. 2005

J Integrative Plant Biology

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Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.

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Section: Gene Cloningmentioning

confidence: 99%

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Zhang

Kai

Lü

et al. 2005

J Integrative Plant Biology

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“…The order of elution was tropinone, tropine, i-tropine, and nicotine. After TR-I and TR-II were completely separated by column chromatography, the enzyme activities were measured by the consumption of NADPH at 340 nm (5,15). The concentration of NADPH in the above reaction mixture was lowered to 0.2 mm to maintain low background absorbance.…”

Section: Plant Materialsmentioning

confidence: 99%

“…An NADPH-dependent oxidoreductase (TR3-I; Fig. 1) that converts tropinone to tropine was first discovered in cultured roots of Datura stramonium (15), but a similar enzyme with different stereospecificity that reduces tropinone to 4I-tropine (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.…”

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confidence: 99%

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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“…A second stereospecific activity, TR II, reduces tropinone to pseudotropine [9]. There was prolonged uncertainty as to whether the reduction of tropinone represented one or two separate prott .,as [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

The formation of 3α‐ and 3β‐acetoxytropanes byDatura stramonium transformed root cultures involves two acetyl‐CoA‐dependent acyltransferases

1991

FEBS Letters

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Tropine (tropan-3~-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in translbrmed root cultures of Datura sfr(mtonit#n. A further acyltransferase activity that acetylates pseudotropine (tropan-3fl-ol) with acetyl-coeuzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in lbrming the observed alkaloid spectrum of D. stramoltium roots discussed.

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Partial Purification and Properties of Tropine Dehydrogenase from Root Cultures ofDatura stramonium

Cited by 33 publications

References 15 publications

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

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

The formation of 3α‐ and 3β‐acetoxytropanes byDatura stramonium transformed root cultures involves two acetyl‐CoA‐dependent acyltransferases

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Partial Purification and Properties of Tropine Dehydrogenase from Root Cultures ofDatura stramonium

Cited by 33 publications

References 15 publications

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

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

The formation of 3α&#x2010; and 3β&#x2010;acetoxytropanes byDatura stramonium transformed root cultures involves two acetyl&#x2010;CoA&#x2010;dependent acyltransferases

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The formation of 3α‐ and 3β‐acetoxytropanes byDatura stramonium transformed root cultures involves two acetyl‐CoA‐dependent acyltransferases