Papaverine (l-Tyrosine-Derived Alkaloid) | SpringerLink

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The biosynthetic origin of some alkaloids derived from the amino acid l ‐tyrosine ..

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Biosynthesis of papaverine - ScienceDirect

Alkaloids are defined as basic compounds derived from amino acids, often with heterocyclically bound nitrogen. Pseudoalkaloids are nitrogen‐containing natural products that do not arise from an amino acid core and acquire their nitrogen by transamination of a precursor derived from other biosynthetic pathways.

Biosynthesis of Papaverine from Tyrosine The metabolism of most alkaloids is poorly known.
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High performance thin layer chromatography (HPTLC) was used for estimating the alkaloid contents in tissue samples. Tissues harvested after 5 hours of wound treatment were dried and ground to fine powder. The powdered samples were soaked overnight in 4 ml methanol and filtered through whattman No.1 filter paper. Extracts were concentrated by drying it in a water bath and re-dissolving in methanol for loading on silica gel 60F254. Standards of Morphine, thebaine, codeine and papaverine were loaded in parallel for comparison. The mobile phase used was toluene: Acetone: Methanol: Ammonia (40:40:8:2). Metabolite analysis was performed using the HPTLC system as described in , . The data represent the mean value ± SD of two independent experiments performed in triplicates. Sanguinarine was extracted from 1g of powdered tissue to prepare acidified methanol (2.0% HCL) extract by hot percolation at 50°C for 1 hour. The extract was filtered, pooled and neutralized with ammonia solution (40%). The pH of the extract was maintained at 9.0. Finally the extract was further fractionated with diethyl ether. The fractions were pooled, concentrated under vacuum and stored at 4°C prior to chromatographic analysis. Sangunarine was estimated using high-performance liquid chromatography–photodiode array detector (HPLC–PDA-MS) system – (Shimadzu Kyoto, Japan).

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BIOSYNTHESIS OF OPIUM ALKALOIDS tyrosine DOPA dopamine norlaudanosoline thebaine codeine morphine papaverine * * * * * * * * …
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A number of transcriptional regulators regulating TIA pathway form C. roseus have been identified and characterized . TIA production is also increased by ectopic expression of genes encoding rate limiting enzymes. Such as overexpression of TDC (tryptophan decarboxylase) gene in C. roseus led to moderate increase in alkaloid accumulation . A more promising approach is the ectopic expression of transcription factors that can regulate multiple steps of the pathway leading to increased accumulation of metabolites . These transcriptional regulators are the site of single step manipulation of metabolic pathways and can be much more effective in metabolic engineering of multiple step biosynthetic pathways . Some of the characterized AP2 like transcription factors positively regulating the TIA pathway are ORCA3, ORCA2 and ORCA1. On the other hand ZCT1, ZCT2 and ZCT3 are the zinc finger repressors of TIA pathway , . WRKY transcription factor families are shown to be involved in alkaloid biosynthesis. The WRKY family proteins contain one or two copies of a DNA-binding domain, designated as WRKY domain, composed of about 50–60 amino acids with the N-terminal conserved motif WRKYGXK and a zinc finger motif (C-X4-8-C-X22-28-H-X1-2-H/C) at the C-terminus . WRKY proteins regulate expression of downstream genes by interacting with the cis-element W-box [TTGAC (T/C)], localized in the promoter regions of the target genes , . WRKY family TFs has shown diversified role in defense, development and metabolism . Recently identified CrWRKY transcription factor from C. roseus activate the TDC gene by directly targeting its promoter . Over expression of CrWRKY in C. roseus hairy root induces the expression of TDC and the ZCT TFs and represses the ORCA2, ORCA3 and CrMYC2 expression . In addition methyl jasmonate inducible WRKY family genes are involved in the production of defense compounds such as flavanoids and terpenoids in Medicago- truncatula.

A subtracted cDNA library was constructed from poly (A+) RNA isolated from eight weeks old seedlings of wounded and control P. somniferum. The whole library was represented by around 1500 recombinant clones. A total of approximately 800 clones were randomly picked, stocked and sequenced using vector specific M13 forward and reverse primers. Out of 800 sequences thus generated, 167 non redundant sequences in total were found to be of good quality, and further submitted to Gen Bank (NCBI). Wound responsive ESTs were validated by hybridizing radio-labelled first strand cDNA probes, using poly (A+) RNA isolated from control and wound stressed samples. Repetitive DNA array hybridization was performed for 167 generated sequences, out of which eighty ESTs were showing more than two fold induction after 5 hours of wounding. Fold expression of ESTs was calculated according to Seki et al. . Effective signal intensities of the spots were calculated after subtracting the background and normalizing it with the intensity of the negative control (NPT II). Fold induction was presented as the expression ratio (wound to control) of each ESTs to that of actin. A list of 80 unique transcripts along with their annotations, average fold inductions, e-values, and standard deviations are presented in . The representative results shown have been repeated thrice with three different sets of wounded cDNA probe to verify the reproducibility. Based on the BLASTX results ESTs (80) were classified into seven different categories such as metabolism (12.2%), transcription (4.8%), cellular transport (7.2%), cell defense (19.5%), cellular organization (11.0%), BIAs Pathway enzymes (11.0%) and unclassified (34.3%) (). There were a number of redundant clones suggesting their abundance in wounded samples. Most notable were NAC transcription factors (5 clones), Dirigent related proteins (8 clones), WRKY transcription factors (7 clones) etc. Wound induced transcripts were subjected to semi-quantitative and real time expression analysis to validate the differential dot-blot hybridization result. Semi-quantitative RT PCR analysis of seven randomly selected ESTs confirming the dot-blot analysis is shown in . In DNA-Array hybridization analysis, methyl transferase genes involved in BIAs biosynthetic pathway, namely 6-OMT (GO238828), 7-OMT (GO238834), and 4-OMT (GO238826) showed maximum induction of 20.34, 12.24 and 8.32 fold respectively (). Other than BIAs pathway transcripts, Unnamed protein (GO238816) showed induction of 6.9 fold followed by induction of 7. 93 fold in dirigent related protein (GO238823).

norlaudanosoline, implying that two

Plant biosynthesis of tyrosine from shikimic acid. In plants and most microorganisms, tyr is produced via prephenate, an intermediate on the shikimate pathway.
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Papaver somniferum is an important medicinal plant. Its medicinal properties are due to benzylisoquinoline alkaloids (BIAs) . BIAs represents approximately 25, 00 elucidated natural product structures found mainly in Papaveraceae, Ranunculaceae, Berberidaceae and Menispermaceae . BIA biosynthesis start with the formation of two L-tyrosine derivatives, 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine (). The synthesis of 4-HPAA and dopamine from tyrosine is catalyzed by tyrosine aminotransferase (TyrAT) and tyrosine/DOPA decarboxylase (TYDC) respectively , . Norcoclaurine synthase (NCS) catalyzes the condensation of 4-HPAA and dopamine yielding (S)-norcoclaurine . Subsequently (S)-methylcoclaurine is synthesized after sequential methylations by norcoclaurine 6-O-methyltransferase (6OMT) and coclaurine N-methyltransferase (CNMT) . (S)-N-Methylcoclaurine-3-hydroxylase (NMCH) catalyzes the 3-hydroxylation of (S)-N-methylcoclaurine to (S)-3-hydroxy-N-methylcoclaurine, which is further converted by 3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (4OMT) to (S)-reticuline , . (S)-reticuline, is the central intermediate from which morphinan alkaloid branch pathway starts with the epimerization of (S)-reticuline to (R)-reticuline by 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR) , . (R)-reticuline is converted to salutaridine by salutaridine synthase (SalSyn) which is subsequently reduced by salutaridine reductase , . Salutaridinol 7-O-acetyltransferase (SalAT) catalyzes the conversion of resulting intermediate to salutaridinol-7-O-acetate ; Salutaridinol-7-O-acetate is further rearranges itself spontaneously or enzymatically to thebaine. Thebaine 6-O- demethylase (T6ODM) and Codeine O-demethylase (CODM) catalyze demethylation of thebaine to oripavine and neopinone . T6ODM further catalyzes conversion of oripavine to morphinone . The NADPH dependent enzyme codeinone reductase (COR) converts (2)-codeinone to (2)-codeine as the penultimate step in the morphine biosynthetic pathway . Most of the enzymes involved in morphinone biosynthetic pathway are characterized, but not much information is known for the branched pathway which leads to formation of noscapine, sanguinarine and papaverine. The Non-Morphinan alkaloid biosynthesis begins with the conversion of (S)-reticuline to (S)-scoulerine, the first committed step catalyzed by berberine bridge enzyme (BBE) , , , . From here the pathway gets diverted for sanguinarine alkaloid biosynthesis initiated by the synthesis of (S)-stylopine, catalyzed by CYPs cheilanthifoline synthase (CheSyn) and stylopine synthase (STSY) , . The subsequent step for the dihydrosanguinarine synthesis involves tetrahydroprotoberberine cis-N-methyltransferase (TNMT), methylstylopine 14-hydroxylase (MSH) and protopine 6-hydroxylase (P6H) , , . Dihydrosanguinarine is oxidized to sanguinarine by dihydrobenzophenanthridine oxidase (DBOX) , . The second diversion from (S)-scoulerine is turned on by Scoulerine 9-O-methyltransferase (SOMT) , , which converts (S)-scoulerine to (S)-tetrahydrocolumbamine. In next step canadinesynthase forms (S)-canadine and subsequently, tetrahydroprotoberberine N-methyltransferase (TNMT) yields N-methylcanadine . The formation of narcotoline occurs, which is O-methylated to noscapine . Palmatine biosynthesis is reported in Coptis japonica, proceeds via columbamine or tetrahydropalmatine involving (S)-tetrahydroxyprotoberberine oxidase (STOX) and columbamine O-methyltransferase (CoOMT) , . Biosynthesis of Papaverine is not well understood, however, two pathways have been proposed. The first one begins with the conversion of (S)-reticuline to (S)-laudanine by reticuline 7-O-methyltransferase (7OMT) , , and as proposed in second it starts at (S)-coclaurine and involve an unique 30-hydroxylase similar to NMCH, norreticuline7-O-methyltransferase (N7OMT). A generalized scheme of the BIAs pathway is shown in .

Real time expression analysis was used to study the expression of PsWRKY transcript under different stress conditions. Real-time primers were made from a PsWRKY CDNA fragment representing the unique C-terminal end of the protein. Very low basal level of PsWRKY transcript was detected under control condition showing its requirement in normal development or metabolic processes. The maximum expression of PsWRKY under control condition was observed in capsule, followed by straw and root, which are the major sites of metabolite synthesis and accumulation in opium poppy (). Under dehydration transcript level of PsWRKY showed maximum induction of 2.5 fold after 3 hours and then decrease in its level after 5 hours (). Its transcript level reached a maximum of more than 6 fold after 1 hour and decreased after 3 hours in case of cold treatment (). Salt stress induced PsWRKY transcript after 1 hour of treatment; then went down after 3 hours and again induced maximally after 5 hours (). Accumulation of PsWRKY in response to wounding showed early induction i.e. just after 1 hour of treatment, its transcript level decreased to 2.5 fold after 3 hours, however it showed a maximum induction of 6 fold after 5 hours (). Methyl jasmonate treatment also induced PsWRKY transcript in a time dependent manner (). After ABA treatment PsWRKY transcript showed maximum accumulation of 2 fold just after 1 hour and it went down to basal level at 3 hours and 5 hours.

The tracer experiments carried out so far on the biosynthesis of papaverine in P
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Wounding is required to be made in the walls of the green seed pod of Opium poppy prior exudation of latex. To withstand this kind of trauma plants regulate expression of some metabolites through an induced transcript level. 167 unique wound-inducible ESTs were identified by a repetitive round of cDNA subtraction after 5 hours of wounding in Papaver somniferum seedlings. Further repetitive reverse northern analysis of these ESTs revealed 80 transcripts showing more than two fold induction, validated through semi-quantitative RT-PCR & real time expression analysis. One of the major classified categories among identified ESTs belonged to benzylisoquinoline transcripts. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to wounding revealed increased accumulation of narcotine and papaverine. Promoter analysis of seven transcripts of BIAs pathway showed the presence of W-box cis-element with the consensus sequence of TGAC, which is the proposed binding site for WRKY type transcription factors. One of the Wound inducible ‘WRKY’ EST isolated from our subtracted library was made full-length and named as ‘PsWRKY’. Bacterially expressed PsWRKY interacted with the W-box element having consensus sequence TTGACT/C present in the promoter region of BIAs biosynthetic pathway genes. PsWRKY further activated the TYDC promoter in yeast and transiently in tobacco BY2 cells. Preferential expression of PsWRKY in straw and capsule and its interaction with consensus W-box element present in BIAs pathway gene transcripts suggest its possible involvement in the wound induced regulation of BIAs pathway.

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