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Benzylisoquinoline Alkaloid

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Peter J. Facchini – 1st expert on this subject based on the ideXlab platform

  • Evidence for the monophyletic evolution of Benzylisoquinoline Alkaloid biosynthesis in angiosperms.
    Phytochemistry, 2020
    Co-Authors: David K Liscombe, Benjamin P Macleod, Natalia Loukanina, Owi I Nandi, Peter J. Facchini

    Abstract:

    Benzylisoquinoline Alkaloids (BIAs) consist of more than 2500 diverse structures largely restricted to the order Ranunculales and the eumagnoliids. However, BIAs also occur in the Rutaceae, Lauraceae, Cornaceae and Nelumbonaceae, and sporadically throughout the order Piperales. Several of these Alkaloids function in the defense of plants against herbivores and pathogens–thus the capacity for BIA biosynthesis is expected to play an important role in the reproductive fitness of certain plants. Biochemical and molecular phylogenetic approaches were used to investigate the evolution of BIA biosynthesis in basal angiosperms. The occurrence of (S)-norcoclaurine synthase (NCS; EC 4.2.1.78) activity in 90 diverse plant species was compared to the distribution of BIAs superimposed onto a molecular phylogeny. These results support the monophyletic origin of BIA biosynthesis prior to the emergence of the eudicots. Phylogenetic analysis of NCS, berberine bridge enzyme and several O-methyltransferases suggest a latent molecular fingerprint for BIA biosynthesis in angiosperms not known to accumulate such Alkaloids. The limited occurrence of BIAs outside the Ranunculales and eumagnoliids suggests the requirement for a highly specialized, yet evolutionarily unstable cellular platform to accommodate or reactivate the pathway in divergent taxa. The molecular cloning and functional characterization of NCS from opium poppy (Papaver somniferum L.) is also reported. Pathogenesis–related (PR)10 and Bet v 1 major allergen proteins share homology with NCS, but recombinant polypeptides were devoid of NCS activity.

  • isolation and characterization of two o methyltransferases involved in Benzylisoquinoline Alkaloid biosynthesis in sacred lotus nelumbo nucifera
    Journal of Biological Chemistry, 2020
    Co-Authors: Ivette M Menendezperdomo, Peter J. Facchini

    Abstract:

    : Benzylisoquinoline Alkaloids (BIAs) are a major class of plant metabolites with many pharmacological benefits. Sacred lotus (Nelumbo nucifera) is an ancient aquatic plant of medicinal value owing to antiviral and immunomodulatory activities linked to its constituent BIAs. Although more than 30 BIAs belonging to the 1-Benzylisoquinoline, aporphine, and bisBenzylisoquinoline structural subclasses and displaying a predominant R enantiomeric conformation have been isolated from N. nucifera, its BIA biosynthetic genes and enzymes remain unknown. Herein, we report the isolation and biochemical characterization of two O-methyltransferases (OMTs) involved in BIA biosynthesis in sacred lotus. Five homologous genes, designated NnOMT1-5 and encoding polypeptides sharing > 40% amino acid sequence identity, were expressed in Escherichia coli Functional characterization of the purified recombinant proteins revealed that NnOMT1 is a regiospecific 1-Benzylisoquinoline 6-O-methyltransferase (6OMT) accepting both R and S substrates, whereas NnOMT5 is mainly a 7-O-methyltransferase (7OMT), with relatively minor 6OMT activity and a strong stereospecific preference for S enantiomers. Available aporphines were not accepted as substrates by either enzyme, suggesting that O-methylation precedes BIA formation from 1-Benzylisoquinoline intermediates. Km values for NnOMT1 and NnOMT5 were 20 μM and 13 μM for (R,S)-norcoclaurine and (S)-N-methylcoclaurine, respectively, similar to those for OMTs from other BIA-producing plants. Organ-based correlations of Alkaloid content, OMT activity in crude extracts, and OMT gene expression supported physiological roles for NnOMT1 and NnOMT5 in BIA metabolism, occurring primarily in young leaves and embryos of sacred lotus. In summary, our work identifies two OMTs involved in BIA metabolism in the medicinal plant N. nucifera.

  • a single residue determines substrate preference in Benzylisoquinoline Alkaloid n methyltransferases
    Phytochemistry, 2020
    Co-Authors: Jeremy S. Morris, Lisa Yu, Peter J. Facchini

    Abstract:

    Abstract N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including Alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of Benzylisoquinoline Alkaloids (BIAs) involves conversion of the secondary amine (S)-coclaurine into the tertiary amine (S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT). Subsequent enzymatic steps yield the core intermediate (S)-reticuline, from which various branch pathways for the biosynthesis of major BIAs such as morphine, noscapine and sanguinarine diverge. An additional N-methylation yielding quaternary BIAs is catalyzed by reticuline N-methyltransferase (RNMT), such as in the branch pathway leading to the taxonomically widespread and ecologically significant Alkaloid magnoflorine. Despite their functional differences, analysis of primary sequence information has been unable to accurately distinguish between CNMT-like and RNMT-like enzymes, necessitating laborious in vitro screening. Furthermore, despite a recent emphasis on structural characterization of BIA NMTs, the features and mechanisms underlying the CNMT-RNMT functional dichotomy were unknown. We report the identification of structural variants tightly correlated with function in known BIA NMTs and show through reciprocal mutagenesis that a single residue acts as a switch between CNMT- and RNMT-like functions. We use yeast in vivo screening to show that this discovery allows for accurate prediction of activity strictly from primary sequence information and, on this basis, improve the annotation of previously reported putative BIA NMTs. Our results highlight the unusually short mutational distance separating ancestral CNMT-like enzymes from more evolutionarily advanced RNMT-like enzymes, and thus help explain the widespread yet sporadic occurrence of quaternary BIAs in plants. While this is the first report of structural variants controlling mono-versus di-methylation activity among plant NMT enzymes, comparison with bacterial MT enzymes also suggests possible convergent evolution.

Fumihiko Sato – 2nd expert on this subject based on the ideXlab platform

  • modulation of Benzylisoquinoline Alkaloid biosynthesis by heterologous expression of cjwrky1 in eschscholzia californica cells
    PLOS ONE, 2017
    Co-Authors: Yasuyuki Yamada, Tomoe Shimada, Yukiya Motomura, Fumihiko Sato

    Abstract:

    : Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole Alkaloids (MIAs), nicotine Alkaloids, and Benzylisoquinoline Alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in Alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.

  • unraveling additional o methylation steps in Benzylisoquinoline Alkaloid biosynthesis in california poppy eschscholzia californica
    Plant and Cell Physiology, 2017
    Co-Authors: Ratmoyo Purwanto, Yasuyuki Yamada, Kentaro Hori, Fumihiko Sato

    Abstract:

    : California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active Benzylisoquinoline Alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple Benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3′-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.

  • tyrosine phosphorylation and protein degradation control the transcriptional activity of wrky involved in Benzylisoquinoline Alkaloid biosynthesis
    Scientific Reports, 2016
    Co-Authors: Yasuyuki Yamada, Fumihiko Sato

    Abstract:

    Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis

Yasuyuki Yamada – 3rd expert on this subject based on the ideXlab platform

  • modulation of Benzylisoquinoline Alkaloid biosynthesis by heterologous expression of cjwrky1 in eschscholzia californica cells
    PLOS ONE, 2017
    Co-Authors: Yasuyuki Yamada, Tomoe Shimada, Yukiya Motomura, Fumihiko Sato

    Abstract:

    : Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole Alkaloids (MIAs), nicotine Alkaloids, and Benzylisoquinoline Alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in Alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.

  • unraveling additional o methylation steps in Benzylisoquinoline Alkaloid biosynthesis in california poppy eschscholzia californica
    Plant and Cell Physiology, 2017
    Co-Authors: Ratmoyo Purwanto, Yasuyuki Yamada, Kentaro Hori, Fumihiko Sato

    Abstract:

    : California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active Benzylisoquinoline Alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple Benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3′-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.

  • tyrosine phosphorylation and protein degradation control the transcriptional activity of wrky involved in Benzylisoquinoline Alkaloid biosynthesis
    Scientific Reports, 2016
    Co-Authors: Yasuyuki Yamada, Fumihiko Sato

    Abstract:

    Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in Benzylisoquinoline Alkaloid biosynthesis