Elucidation of the (R)-enantiospecific benzylisoquinoline alkaloid biosynthetic pathways in sacred lotus (Nelumbo nucifera).

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant specialized metabolites found mainly in members of the order Ranunculales, including opium poppy (Papaver somniferum), for which BIA biosynthetic pathways leading to the critical drugs morphine, noscapine, and sanguinarine have been elucidated. Sacred lotus (Nelumbo nucifera), in the order Proteales, accumulates medicinal BIAs in the proaporphine, aporphine, and bisbenzylisoquinoline structural subgroups with a prevalence of R enantiomers, opposed to the dominant S configuration occurring in the Ranunculales. Nevertheless, distinctive BIA biosynthetic routes in sacred lotus have not been explored. In planta labeling experiments and in vitro assays with recombinant enzymes and plant protein extracts showed that dopamine and 4-hydroxyphenylacetaldehyde derived from L-tyrosine serve as precursors for the formation of (R,S)-norcoclaurine in sacred lotus, whereas only (R)-norcoclaurine byproducts are favored in the plant by action of R-enantiospecific methyltransferases and cytochrome P450 oxidoreductases (CYPs). Enzymes responsible for the R-enantiospecific formation of proaporphine (NnCYP80Q1) and bisbenzylisoquinoline (NnCYP80Q2) scaffolds, and a methylenedioxy bridge introduction on aporphine substrates (NnCYP719A22) were identified, whereas additional aspects of the biosynthetic pathways leading to the distinctive alkaloid profile are discussed. This work expands the availability of molecular tools that can be deployed in synthetic biology platforms for the production of high-value alkaloids.

Benzylisoquinoline alkaloid analysis using high-resolution Orbitrap LC-MSn.

Benzylisoquinoline alkaloids (BIAs) have profound implications on human health owing to their potent pharmacological properties. Notable naturally occurring BIAs are the narcotic analgesics morphine, the cough suppressant codeine, the potential anticancer drug noscapine, the muscle relaxant papaverine, and the antimicrobial sanguinarine, all of which are produced in opium poppy (Papaver somniferum). Thebaine, an intermediate in the biosynthesis of codeine and morphine, is used in the manufacture of semisynthetic opiates, including oxycodone and naloxone. As the only commercial source of pharmaceutical opiates, opium poppy has been the focus of considerable research to understand BIA metabolism in the plant. The elucidation of several BIA biosynthetic pathways has enabled the development of synthetic biology platforms aimed at the alternative commercial production of valuable phytochemicals in microorganisms. The detection and identification of BIA pathway products and intermediates in complex extracts is essential for the continuing advancement of research in plant specialized metabolism and microbial synthetic biology. Herein, we report the use of liquid chromatography coupled with linear trap quadrupole and high-resolution Orbitrap multistage mass spectrometry to characterize 44 authentic BIAs using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and pulsed Q collision-induced dissociation (PQD) MS2 fragmentation, with MS2 CID followed by MS3 and MS4 fragmentation. Our deep library of diagnostic spectral data constitutes a valuable resource for BIAs identification. In addition, we identified 22 BIAs in opium poppy latex and roots extracts.

Isolation and characterization of two O-methyltransferases involved in benzylisoquinoline alkaloid biosynthesis in sacred lotus (Nelumbo nucifera).

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 because of 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 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.

Benzylisoquinoline Alkaloids Biosynthesis in Sacred Lotus.

Sacred lotus (Nelumbo nucifera Gaertn.) is an ancient aquatic plant used throughout Asia for its nutritional and medicinal properties. Benzylisoquinoline alkaloids (BIAs), mostly within the aporphine and bisbenzylisoquinoline structural categories, are among the main bioactive constituents in the plant. The alkaloids of sacred lotus exhibit promising anti-cancer, anti-arrhythmic, anti-HIV, and anti-malarial properties. Despite their pharmacological significance, BIA metabolism in this non-model plant has not been extensively investigated. In this review, we examine the diversity of BIAs in sacred lotus, with an emphasis on the distinctive stereochemistry of alkaloids found in this species. Additionally, we discuss our current understanding of the biosynthetic genes and enzymes involved in the formation of 1-benzylisoquinoline, aporphine, and bisbenzylisoquinoline alkaloids in the plant. We conclude that a comprehensive functional characterization of alkaloid biosynthetic enzymes using both in vitro and in vivo methods is required to advance our limited knowledge of BIA metabolism in the sacred lotus.

Cuban flora species as a potential source of DNA protective compounds / Especies de la flora cubana cómo fuente potencial de compuestos protectores del DNA

Environmental exposure to genotoxic agents represents a major health concern for modern society. DNA damage could lead to mutations, which accumulative effect is closely related to degenerative and lethal diseases, such as cancer. Because of their structural and chemical diversity natural products play a fundamental role in pharmaceutical sciences for novel drug discovery. The present review article focuses on pre-clinical studies done with some species from Cuban flora that have been tested with positive antigenotoxic properties against different genotoxins. Special emphasis regarding molecular mechanisms suggested, from antioxidant activity to DNA repair modulation, a critical discussion of the state of art and the perspectives in the use of these plants as a new and promising strategy for genoprotection in the 21st Century are included.

La exposición ambiental a agentes genotóxicos representa un problema de salud significativo en la sociedad actual. El daño al ADN puede generar mutaciones, cuyo efecto acumulativo se encuentra estrechamente relacionado con enfermedades degenerativas y letales como el cáncer. Debido a su diversidad estructural y química los productos naturales juegan un papel fundamental en las ciencias farmacéuticas en el descubrimiento de nuevas drogas. El presente artículo de revisión puntualiza estudios pre-clínicos realizados con determinadas especies de la flora cubana que han sido estudiadas con una respuesta antioxidante positiva frente a diferentes genotoxinas. Se enfatizan especialmente los mecanismos moleculares sugeridos, desde actividad antioxidante hasta modulación de la reparación del ADN, así como una discusión crítica del estado del arte y las perspectivas en el empleo de estas plantas como una estrategia nueva y prometedora para la genoprotección en el siglo 21.