Lineage-Specific CYP80 Expansion and Benzylisoquinoline Alkaloid Diversity in Early-Diverging Eudicots.

Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.

Antidepressant effects of total alkaloids of Fibraurea recisa on improving corticosterone-induced apoptosis of HT-22 cells and chronic unpredictable mild stress-induced depressive-like behaviour in mice.

CONTEXT: Fibraurea recisa Pierre. (Menispermaceae) (FR) is a traditional Chinese medicine known as "Huangteng." The total alkaloids of FR (AFR) are the main active ingredients. However, the pharmacological effects of AFR in the treatment of depression have not been reported. OBJECTIVES: This study investigates the antidepressant effects of AFR by network pharmacology and verification experiments. MATERIALS AND METHODS: Compound-Target-Pathway (C-P-T) network of FR and depression was constructed through network pharmacology. In vitro, HT-22 cells were treated with corticosterone (CORT) solution (0.35 mg/mL), then AFR (0.05 mg/mL) solution and inhibitor AZD6244 (14 µM/mL) or BAY11-7082 (10 µM/mL) were added, respectively. The cell viability was detected by CCK-8. In vivo, C57BL/6 mice were divided into 5 groups, namely the normal group, the CUMS group, the AFR (400 mg/kg) group, and the 2 groups that were simultaneously administered the inhibitory group AZD6244 (8 mg/kg) and BAY11-7082 (5 mg/kg). Western blotting was used to assess the expression level of the proteins. RESULTS: AFR could protect HT-22 cells from CORT-induced damage and increase the cell viability from 49.12 ± 3.4% to 87.26 ± 1.5%. Moreover, AFR significantly increased the levels of BDNF (1.3, 1.4-fold), p-ERK (1.4, 1.2-fold) and p-CERB (1.6, 1.3-fold), and decreased the levels of NLRP3 (11.3%, 31.6%), ASC (19.2%, 34.2%) and caspase-1 (18.0%, 27.6%) in HT-22 cells and the hippocampus, respectively. DISCUSSION AND CONCLUSIONS: AFR can improve depressive-like behaviours and can develop drugs for depression treatment. Further studies are needed to validate its potential in clinical medicine.

The chloroalkaloid (-)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants.

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.

Exploring Secondary Metabolites Database of Apocynaceae, Menispermaceae, and Annonaceae to Select Potential Anti-HCV Compounds.

BACKGROUND: Hepatitis C is a disease that constitutes a serious global health problem, is often asymptomatic and difficult to diagnose and about 60-80% of infected patients develop chronic diseases over time. As there is no vaccine against hepatitis C virus (HCV), developing new cheap treatments is a big challenge. OBJECTIVE: The search for new drugs from natural products has been outstanding in recent years. The aim of this study was to combine structure-based and ligand-based virtual screening (VS) techniques to select potentially active molecules against four HCV target proteins from in-house secondary metabolite dataset (SistematX). MATERIALS AND METHODS: From the ChEMBL database, we selected four sets of 1199, 355, 290 and 237chemical structures with inhibitory activity against different targets of HCV to create random forest models with an accuracy value higher than 82% for cross-validation and test sets. Afterward, a ligandbased virtual screen of the entire 1848 secondary metabolites database stored in SistematX was performed. In addition, a structure-based virtual screening was also performed for the same set of secondary metabolites using molecular docking. RESULTS: Finally, using consensus analyses approach combining ligand-based and structure-based VS, three alkaloids were selected as potential anti-HCV compounds. CONCLUSION: The selected structures are a starting point for further studies in order to develop new anti- HCV compounds based on natural products.

Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the ß-sheet.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.

Metabolome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BACKGROUND: Recent progress toward the elucidation of benzylisoquinoline alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties. RESULTS: We applied a multi-platform approach incorporating four different analytical methods to examine 20 non-model, BIA-accumulating plant species. Plants representing four families in the Ranunculales were chosen based on reported BIA content, taxonomic distribution and importance in modern/traditional medicine. One-dimensional (1)H NMR-based profiling quantified 91 metabolites and revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content. Mono- and disaccharide sugars were generally lower in roots and rhizomes compared with stems, and a variety of metabolites distinguished callus tissue from intact plant organs. Direct flow infusion tandem mass spectrometry provided a broad survey of 110 lipid derivatives including phosphatidylcholines and acylcarnitines, and high-performance liquid chromatography coupled with UV detection quantified 15 phenolic compounds including flavonoids, benzoic acid derivatives and hydroxycinnamic acids. Ultra-performance liquid chromatography coupled with high-resolution Fourier transform mass spectrometry generated extensive mass lists for all species, which were mined for metabolites putatively corresponding to BIAs. Different alkaloids profiles, including both ubiquitous and potentially rare compounds, were observed. CONCLUSIONS: Extensive metabolite profiling combining multiple analytical platforms enabled a more complete picture of overall metabolism occurring in selected plant species. This study represents the first time a metabolomics approach has been applied to most of these species, despite their importance in modern and traditional medicine. Coupled with genomics data, these metabolomics resources serve as a key resource for the investigation of BIA biosynthesis in non-model plant species.

Tiliacora triandra, an Anti-Intoxication Plant, Improves Memory Impairment, Neurodegeneration, Cholinergic Function, and Oxidative Stress in Hippocampus of Ethanol Dependence Rats.

Oxidative stress plays an important role in brain dysfunctions induced by alcohol. Since less therapeutic agent against cognitive deficit and brain damage induced by chronic alcohol consumption is less available, we aimed to assess the effect of Tiliacora triandra extract, a plant possessing antioxidant activity, on memory impairment, neuron density, cholinergic function, and oxidative stress in hippocampus of alcoholic rats. Male Wistar rats were induced ethanol dependence condition by semivoluntary intake of alcohol for 15 weeks. Alcoholic rats were orally given T. triandra at doses of 100, 200, and 400 mg·kg(-1)BW for 14 days. Memory assessment was performed every 7 days while neuron density, activities of AChE, SOD, CAT, and GSH-Px and, MDA level in hippocampus were assessed at the end of study. Interestingly, the extract mitigated the increased escape latency, AChE and MDA level. The extract also mitigated the decreased retention time, SOD, CAT, and GSH-Px activities, and neurons density in hippocampus induced by alcohol. These data suggested that the extract improved memory deficit in alcoholic rats partly via the decreased oxidative stress and the suppression of AChE. Therefore, T. triandra is the potential reagent for treating brain dysfunction induced by alcohol. However, further researches are necessary to understand the detail mechanism and possible active ingredient.

Conversion of dechlorodauricumine into chlorinated alkaloids in Menispermum dauricum root culture.

(15)N-Labeled dechlorodauricumine and dechloroacutumine were isolated from Menispermum dauricum roots cultured in a chloride-deficient medium, in which nitrogen-containing macro-components K(14)NO(3) and ((14)NH(4))(2)SO(4) were replaced by K(15)NO(3) and ((15)NH(4))(2)SO(4), respectively. These (15)N-labeled substrates were supplied independently to the roots cultured in a chloride-enriched medium. LC-ESI-MS analysis of alkaloids extracted from the roots, harvested 5 and 10 days after administering the (15)N-labeled substrates, revealed that the (15)N derived from dechlorodauricumine was much more effectively incorporated into chlorinated alkaloids than that derived from dechloroacutumine. These findings suggest that dechlorodauricumine is the principal precursor of the chlorinated alkaloids produced by M. dauricum roots.