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Peter J Facchini - One of the best experts on this subject based on the ideXlab platform.
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purine permease type benzylisoquinoline alkaloid transporters in opium poppy
Plant Physiology, 2019Co-Authors: Mehran Dastmalchi, Limei Chang, Rongji Chen, Lisa Yu, Jillian M Hagel, Peter J Facchini, Xue Chen, Peter J FacchiniAbstract:Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.
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heterodimeric o methyltransferases involved in the biosynthesis of noscapine in opium poppy
Plant Journal, 2018Co-Authors: Myung Ryeol Park, Xue Chen, Dean E Lang, Peter J FacchiniAbstract:Noscapine biosynthesis in opium poppy involves three characterized O-methyltransferases (OMTs) and a fourth responsible for the 4'-methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes (OMT2 and OMT3). Neither OMT2 nor OMT3 form stable homodimers, but yield a substrate-specific heterodimer when their genes are co-expressed in Escherichia coli. The only substrate, 4'-O-desmethyl-3-O-acetylpapaveroxine, is a seco-berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4'-O-methylation leading to the formation of narcotine hemiacetal. In the absence of 4'-O-methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4'-hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1-hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6OMT, which shares 81% amino acid sequence identity with OMT3, also formed a functionally similar heterodimer with OMT2. Suppression of OMT2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT2:OMT3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate-binding domain, although site-directed mutagenesis revealed OMT2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.
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systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy
Plant Journal, 2012Co-Authors: Isabel Desgagnepenix, Peter J Facchini, Peter J FacchiniAbstract:SUMMARY Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3¢-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3¢hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3¢-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine.
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mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis an enzymatic pictet spengler reaction
Biochemistry, 2007Co-Authors: Shannon Bunn, Peter J Facchini, David K. Liscombe, Peter J Facchini, Martin E TannerAbstract:Norcoclaurine synthase catalyzes an asymmetric Pictet−Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a k...
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the role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy
Plant Journal, 2006Co-Authors: Nailish Samanani, Joenel Alcantara, Richard Bourgault, Katherine G Zulak, Peter J Facchini, Nailish Samanani, Peter J FacchiniAbstract:The benzylisoquinoline alkaloids of opium poppy, including the narcotic analgesics morphine and codeine, accumulate in the multinucleate cytoplasm of specialized laticifers that accompany vascular tissues throughout the plant. In mature opium poppy plants, immunofluorescence labeling using specific antibodies showed that four alkaloid biosynthetic enzymes, (S)-norcoclaurine 6-O-methyltransferase (6OMT), (S)-coclaurine N-methyltransferase (CNMT), (S)-3'-hydroxy-N-methylcoclaurine-4'-O-methyltransferase (4'OMT) and salutaridinol-7-O-acetyltransferase (SAT) were restricted to sieve elements of the phloem adjacent or proximal to laticifers. The identity of sieve elements was confirmed by (i) the specific immunogold labeling of the characteristic cytoplasm of this cell type, (ii) the co-localization of a sieve element-specific H(+)-ATPase with all biosynthetic enzymes and (iii) the strict association of sieve plates with immunofluorescent cells. The localization of laticifers was demonstrated antibodies specific to major latex protein (MLP), which is characteristic of this cell type. In situ hybridization using antisense RNA probes for 6OMT, CNMT, 4'OMT and SAT showed that the corresponding gene transcripts were found in the companion cell paired with each sieve element. Seven benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1), berberine bridge enzyme, codeinone reductase, 6OMT, CNMT, 4'OMT and SAT were localized by immunofluorescence labeling to the sieve elements in the root and hypocotyl of opium poppy seedlings. The abundance of these enzymes increased rapidly between 1 and 3 days after seed germination. The localization of seven biosynthetic enzymes to the sieve elements provides strong support for the unique, cell type-specific biosynthesis of benzylisoquinoline alkaloids in the opium poppy.
Fumihiko Sato - One of the best experts on this subject based on the ideXlab platform.
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BIA biosynthetic pathways in BIA-producing plants.
2017Co-Authors: Yasuyuki Yamada, Tomoe Shimada, Yukiya Motomura, Fumihiko SatoAbstract:Black and gray letters show biosynthetic enzymes in C. japonica and E. californica, respectively. Broken lines indicate uncharacterized enzyme reactions. NCS, (S)-norcoclaurine synthase; 6OMT, (S)-norcoclaurine 6-O-methyltransferase; CNMT, (S)-coclaurine-N-methyltransferase; CYP80B1, (S)-N-methylcoclaurine 3’-hydroxylase; 4’OMT, (S)-3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase; BBE, berberine bridge enzyme; SMT, (S)-scoulerine 9-O-methyltransferase of C. japonica; CYP719A1, (S)-canadine synthase; THBO, (S)-tetrahydroprotoberberine oxidase; CYP719A5, (S)-cheilanthifoline synthase; CYP719A2/3, (S)-stylopine synthase; TNMT, (S)-tetrahydroprotoberberine cis-N-methyltransferase, MSH, (S)-N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase; SR, sanguinarine reductase; G3OMT, (S)-scoulerine 9-O-methyltransferase of California poppy; G11OMT, putative 10-hydroxydihydrosanguinarine 10-O-methyltransferase. CYP1 and CYP2 are putative dihydrobenzophenanthridine 10-hydroxylases.
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Asymmetric synthesis of tetrahydroisoquinolines by enzymatic Pictet-Spengler reaction.
Bioscience biotechnology and biochemistry, 2014Co-Authors: Masakatsu Nishihachijo, Hiromichi Minami, Yoshinori Hirai, Shigeru Kawano, Akira Nishiyama, Takane Katayama, Yoshihiko Yasohara, Fumihiko Sato, Hidehiko KumagaiAbstract:Norcoclaurine synthase (NCS) catalyzes the stereoselective Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde as the first step of benzylisoquinoline alkaloid synthesis in plants. Recent studies suggested that NCS shows relatively relaxed substrate specificity toward aldehydes, and thus, the enzyme can serve as a tool to synthesize unnatural, optically active tetrahydroisoquinolines. In this study, using an N-terminally truncated NCS from Coptis japonica expressed in Escherichia coli, we examined the aldehyde substrate specificity of the enzyme. Herein, we demonstrate the versatility of the enzyme by synthesizing 6,7-dihydroxy-1-phenethyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-propyl-1,2,3,4-tetrahydroisoquinoline in molar yields of 86.0 and 99.6% and in enantiomer excess of 95.3 and 98.0%, respectively. The results revealed the enzyme is a promising catalyst that functions to stereoselectively produce various 1-substituted-1,2,3,4-tetrahydroisoquinolines.
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functional analysis of norcoclaurine synthase in coptis japonica
Journal of Biological Chemistry, 2007Co-Authors: Hiromichi Minami, Kinuko Iwasa, Emilyn G Dubouzet, Fumihiko SatoAbstract:Abstract (S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe2+-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.
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molecular characterization of the s adenosyl l methionine 3 hydroxy n methylcoclaurine 4 o methyltransferase involved in isoquinoline alkaloid biosynthesis in coptis japonica
Journal of Biological Chemistry, 2000Co-Authors: Takashi Morishige, Yasuyuki Yamada, Tetsuya Tsujita, Fumihiko SatoAbstract:Abstract S-Adenosyl-l-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′-OMT) catalyzes the conversion of 3′-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, anotherO-methyltransferase,S-adenosyl-l-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4′-OMT preparation from cultured Coptis japonicacells. Heterologously expressed proteins had 6-OMT or 4′-OMT activities, indicative that each cDNA encodes a different enzyme. 4′-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe2+, Cu2+, Co2+, Zn2+, or Ni2+, but none by the SH reagent. 4′-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, anN-methylated substrate, was a much better substrate for 4′-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4′-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two relatedO-methyltransferases is discussed.
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purification and characterization of s adenosyl l methionine norcoclaurine 6 o methyltransferase from cultured coptis japonica cells
FEBS Journal, 1994Co-Authors: Fumihiko Sato, Yasuyuki Katagiri, Tetsuya Tsujita, Shigeru Yoshida, Yasuyuki YamadaAbstract:S-adenosyl-l-methionine:norcoclaurine 6-O-methyltransferase (norcoclaurine 6-O-methyltransferase), which catalyzes the transfer of the S-methyl group of S-adenosyl-l-methionine to the 6-hydroxyl group of 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl)methyl]-6,7-isoquinolinediol (norcoclaurine), was purified from cultured Coptis japonica cells and its enzymic properties were characterized. Purified norcoclaurine 6-O-methyltransferase had apparent pI 4.7, a native molecular mass of 95 kDa (determined by gel filtration) and subunit molecular mass of 40 kDa (SDS/PAGE). The enzyme did not require a divalent cation for activity, and the addition of Fe2+, Cu2+, Co2+, Zn2+, Mn2+, or Ni2+ at 5 mM severely inhibited enzyme activity. Neither p-chloromercuribenzoate, N-methylmaleimide nor iodoacetamide inhibited enzyme activity at 1 mM. 5, 6-Dihydro-9, 10-dime-thoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium (berberine, the end-product of the biosynthetic pathway in which norcoclaurine 6-O-methyltransferase catalyzes an intermediate step) also inhibited the activity by 50% at 10 mM. Norcoclaurine 6-O-methyltransferase methylated both (S)-norcoclaurine and (R)-norcoclaurine and (R,S)-norlaudanosoline. Further characterization of substrate-saturation kinetics and product inhibition of the purified enzyme indicated that norcoclaurine 6-O-methyltransferase follows a bi-bi ping-pong mechanism with Km, values of 2.23 mM and 3.95 mM for (R,S)-norlaudanosoline and S-adenosyl-l-methionine, respectively, while Ki values for S-adenosyl-homocysteine versus S-adenosyl-l-methionine and (R,S)-norlaudanosoline were 2.1 mM and 0.18 mM, respectively.
Meinhart H. Zenk - One of the best experts on this subject based on the ideXlab platform.
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the biosynthesis of papaverine proceeds via s reticuline
Phytochemistry, 2010Co-Authors: Marc Lamshoft, Nadja Grobe, Anthony John Fist, Michael Spiteller, Toni M. Kutchan, Meinhart H. ZenkAbstract:Abstract Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O -methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo . The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into ( S )-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of ( S )-reticuline to generate ( S )-laudanine. A second methylation at the 3′ position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N -demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine.
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1 2 dehydroreticuline synthase the branch point enzyme opening the morphinan biosynthetic pathway
Phytochemistry, 2004Co-Authors: Kazumasa Hirata, Chotima Poeaknapo, J Schmidt, Meinhart H. ZenkAbstract:Abstract A synthase which oxidizes ( S )-reticuline to 1,2-dehydroreticuline has been found to occur in seedlings of opium poppy ( Papaver somniferum L. ). Due to its instability, this enzyme could only be partly purified (ca. 5-fold enrichment). Partial characterization at this stage of purification showed that it does not need a redox cofactor and accepts both ( S )-reticuline and ( S )-norreticuline as substrates. [1- 2 H, 13 C]-( R,S )-reticuline was enzymatically converted into [1- 13 C]-dehydroreticuline, which has been identified by mass spectrometry. Release of the hydrogen atom in position C-1 of the isoquinoline alkaloid during the oxidative conversion, was exploited as a sensitive assay system for this enzyme. The enzyme has a pH optimum of 8.75, a temperature optimum of 37 °C and the apparent K M value for the substrate reticuline was shown to be 117 μM. Moreover it could be demonstrated by sucrose density gradient centrifugation that the enzyme is located in vesicles of varying size. In combination with the previously discovered strictly stereoselective and NADPH dependent 1,2-dehydroreticuline reductase the detection of this enzyme, the 1,2-dehydroreticuline synthase, provides the necessary inversion of configuration and completes the pathway from two molecules of L-tyrosine via ( S )-norcoclaurine to ( R )-reticuline in opium poppy involving a total number of 11 enzymes.
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the norcoclaurine pathway is operative in berberine biosynthesis in coptis japonica
Planta Medica, 1992Co-Authors: Martin J Muller, Meinhart H. ZenkAbstract:The commercially used cell suspension culture of COPTIS JAPONICA Makino (Ranunculaceae) incorporates [beta- (13)C]tyrosine into both halves of the berberine molecule (isoquinoline and benzyl portion) while L-[3'- (18)O]DOPA only labels the isoquinoline portion of this molecule. This labelling pattern indicates that different C (6)-C (2) units derived from tyrosine form this molecule. This result and the incorporation of ( S)-[1- (13)C] norcoclaurine into berberine demonstrates that the biosynthesis of protoberberine alkaloids in COPTIS involves the norcoclaurine but not the norlaudanosoline pathway.
Brijesh Kumar - One of the best experts on this subject based on the ideXlab platform.
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analysis of phytochemical variations in dioecious tinospora cordifolia stems using hplc qtof ms ms and uplc qqqlit ms ms
Phytochemical Analysis, 2016Co-Authors: Vikas Bajpai, Awantika Singh, Preeti Chandra, M P S Negi, Nikhil Kumar, Brijesh KumarAbstract:Introduction The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. Objective To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. Methods Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQLIT-MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. Results Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. Conclusions Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers. Copyright © 2015 John Wiley & Sons, Ltd.
Takashi Morishige - One of the best experts on this subject based on the ideXlab platform.
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overexpression of coptis japonica norcoclaurine 6 o methyltransferase overcomes the rate limiting step in benzylisoquinoline alkaloid biosynthesis in cultured eschscholzia californica
Plant and Cell Physiology, 2007Co-Authors: Takayuki Inui, Nanae Fujii, Kenichi Tamura, Takashi MorishigeAbstract:Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3 0 -hydroxy-N-methylcoclaurine 4 0 -O-methyltransferase (4 0 OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4 0 OMT transformants. HPLC/ liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. japonica 4 0 OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.
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molecular characterization of the s adenosyl l methionine 3 hydroxy n methylcoclaurine 4 o methyltransferase involved in isoquinoline alkaloid biosynthesis in coptis japonica
Journal of Biological Chemistry, 2000Co-Authors: Takashi Morishige, Yasuyuki Yamada, Tetsuya Tsujita, Fumihiko SatoAbstract:Abstract S-Adenosyl-l-methionine:3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′-OMT) catalyzes the conversion of 3′-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, anotherO-methyltransferase,S-adenosyl-l-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4′-OMT preparation from cultured Coptis japonicacells. Heterologously expressed proteins had 6-OMT or 4′-OMT activities, indicative that each cDNA encodes a different enzyme. 4′-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe2+, Cu2+, Co2+, Zn2+, or Ni2+, but none by the SH reagent. 4′-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R,S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, anN-methylated substrate, was a much better substrate for 4′-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4′-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two relatedO-methyltransferases is discussed.
