Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-(13)C]3',4'-dimethoxycinnamic acid, [2-(13)C]3',4'-methylenedioxycinnamic acid (MDCA), [2-(13)C]3',4',5'-trimethoxycinnamic acid, [2-(13)C]sinapic acid, [2-(13)C]- and [2,3-(13)C(2)]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/wo.1007/s00425-002-0834-1.

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“…The rate of 13 C enrichment was calculated from the areas under the M+1, M+2 and M+4 ions (as described by Robins et al 1994). …”

Section: Quantification Of Incorporationmentioning

confidence: 99%

Biosynthesis of podophyllotoxin in Linum album cell cultures

Seidel

Windhövel

Eaton

et al. 2002

Planta

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“…Moreover, 1‐O‐β‐glucose esters can be produced by UDP‐glycosyltransferase (UGT) using specific substrates and UDP‐glucose (Schulenburg et al , ; Zhao et al , ). Previous research identified A. belladonna enzymes responsible for producing acetylpseudotropine, tigloylpseudotropine, and phenylacetylpseudotropine, but not for acetyltropine or phenylacetyltropine (Robins et al , ), suggesting that BAHD acyltransferases, taking CoA activated compounds, participate in acyl modification of pseudotropine but not tropine. Thus, glycosylated phenyllactate (phenyllactylglucose) might be the acyl donor for tropine, and tropine and phenyllactylglucose might condense to littorine when catalysed by serine carboxypeptidase (SCP)‐like acyltransferases (SCPL‐ATs), which catalyse esterification between specific compounds and 1‐O‐β‐glucose esters (Li & Steffens, ).…”

Section: Introductionmentioning

confidence: 97%

Functional genomics analysis reveals two novel genes required for littorine biosynthesis

et al. 2019

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Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood.Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS).UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added.This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

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“…In contrast, tropinone reductase I reduces tropinone to tropine, which leads to the biosynthesis of hyoscyamine and scopolamine. Tropine is then condensed with a phenyllactic acid moiety to form littorine, which undergoes P450-mediated rearrangement to hyoscyamine aldehyde (Robins et al, 1994;Li et al, 2006;O'Connor, 2010). An uncharacterized alcohol dehydrogenase is proposed to convert hyoscyamine aldehyde to hyoscyamine, which is subsequently converted to scopolamine in a two-step epoxidation reaction catalyzed by hyoscyamine 6-hydroxylase (H6H) (Matsuda et al, 1991;Hashimoto et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

et al. 2014

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The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

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Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

Cited by 43 publications

References 32 publications

Biosynthesis of podophyllotoxin in Linum album cell cultures

Biosynthesis of podophyllotoxin in Linum album cell cultures

Functional genomics analysis reveals two novel genes required for littorine biosynthesis

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

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