An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

Simultaneous determination of total ergot alkaloids in wheat flour by Orbitrap mass spectrometry.

The optimization screening methods for total ergot alkaloids in wheat extracts involve transforming them into a single compound, which is then analyzed via high-resolution Orbitrap mass spectrometry (Orbitrap MS). Orbitrap MS provides highly sensitive and accurate mass measurements, enhancing the selectivity and sensitivity of the analysis. Various hydrolysis and reduction methods have been investigated, and the use of superhydrides has emerged as the most effective method for transforming ergopeptine alkaloids. This study also focused on the epimerization of ergot alkaloids, particularly the differences between R- and S-epimers and their impact on the mass spectra. We validated our method by assessing the linearity, sensitivity, recovery, matrix effects, repeatability, and stability. The limits of detection and quantitation were set at 0.43 and 1.30 µg LSA/kg wheat, respectively. The proposed method offers a robust analytical approach for screening and quantifying total ergot alkaloids in wheat samples, addressing important concerns about their presence in food and feed.

Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Derivation of the multiply-branched ergot alkaloid pathway of fungi.

Ergot alkaloids are a large family of fungal specialized metabolites that are important as toxins in agriculture and as the foundation of powerful pharmaceuticals. Fungi from several lineages and diverse ecological niches produce ergot alkaloids from at least one of several branches of the ergot alkaloid pathway. The biochemical and genetic bases for the different branches have been established and are summarized briefly herein. Several pathway branches overlap among fungal lineages and ecological niches, indicating activities of ergot alkaloids benefit fungi in different environments and conditions. Understanding the functions of the multiple genes in each branch of the pathway allows researchers to parse the abundant genomic sequence data available in public databases in order to assess the ergot alkaloid biosynthesis capacity of previously unexplored fungi. Moreover, the characterization of the genes involved in the various branches provides opportunities and resources for the biotechnological manipulation of ergot alkaloids for experimentation and pharmaceutical development.

A Short Synthesis of Ergot Alkaloids and Evaluation of the 5-HT1/2 Receptor Selectivity of Lysergols and Isolysergols.

Key transformations in a four-step synthesis of the ergot alkaloid scaffold include a novel cesium carbonate-mediated hydrogen autotransfer alkylation to generate the C(3)-C(4) bond and an intramolecular Heck reaction that directly establishes the C(9)-C(10) alkene of methyl lysergate. An ester reduction and a streamlined experimental procedure establish a readily scalable, expedient total synthesis of all four stereoisomers of lysergol and isolysergol, including the previously unknown (-)-lysergol, for pharmacological evaluation at 5-HT1A and 5HT2A,B,C receptors. A bicyclic scaffold is also characterized for the first time in the intramolecular Heck coupling.

Sustained vascular contractile response induced by an R- and S-epimer of the ergot alkaloid ergocristine and attenuation by a noncompetitive antagonist.

Vasoconstriction is a known effect associated with ergot alkaloid consumption. The vascular contractile responses are often sustained for an extended period after exposure. Ergot alkaloids exist in two molecular configurations, the C-8-(R)-isomer (R-epimer) and the C-8-(S)-isomer (S-epimer). The sustained vascular contractile response to the R-epimers has been studied previously, unlike the S-epimers which are thought to be biologically inactive. Additionally, antagonists have been utilized to attenuate the vascular contraction associated with the R-epimers of ergot alkaloids utilizing ex vivo techniques. This study utilized an arterial tissue bath to examine and compare the sustained vascular contractile response attributed to ergocristine (R) and ergocristinine (S) using dissected bovine metatarsal arteries. The contractile blocking effect of a noncompetitive alpha-adrenergic antagonist, phenoxybenzamine (POB), was also investigated in precontracted arteries. Arteries (n = 6/epimer) were exposed to a single dose of ergocristine or ergocristinine (1 × 10-6 M in buffer). Each of the epimer doses was followed by a POB (1 × 10-3 M) or methanol (control) treatment at 90 min and the response was observed for another 90 min. Both epimers produced a sustained contractile response over the 180-min incubation period in the control groups. The R-epimer caused a greater sustained contractile response from 60 to 180 min post epimer exposure, compared to the S-epimer (P < 0.05, generalized estimating equations, independent t-test). Phenoxybenzamine caused a decrease in the contractile response induced by ergocristine and ergocristinine from 105 to 180 min, compared to the control (P < 0.05, generalized estimating equations, paired t-test). Overall, these results demonstrate the presence of a sustained vascular contractile response attributed to the R- and S-epimer of an ergot alkaloid with differences in contractile response between the epimers, suggesting differences in receptor binding mechanisms. Furthermore, this study demonstrated that a noncompetitive antagonist could attenuate the sustained arterial contractile effects of both ergot configurations ex vivo. Additional investigation into S-epimers of ergot alkaloids is needed. This research contributes to the understanding of the ergot epimer-vascular receptor binding mechanisms, which may support the investigation of different approaches of minimizing ergot toxicity in livestock.

Ergot alkaloids cause blood vessels to contract when contaminated feed is consumed by animals. Vascular contraction often remains for a prolonged period and involves the binding of ergot to specific receptors in the blood vessels. This study assessed and compared the sustained contraction of cow arteries after exposure to two forms of an ergot alkaloid, namely, ergocristine and ergocristinine. The effects of a specific receptor blocker, phenoxybenzamine, on the vascular contraction induced by these forms were also examined. This study showed that both forms of ergot caused a sustained contraction of cow arteries but to different magnitudes. Differences in contraction could be related to differences in how each form of ergot binds to receptors. The receptor blocker decreased the sustained contractile response of both forms of ergot. Further understanding of how the different forms of ergot bind to receptors, and how to decrease the adverse effects, may help mitigate the toxic effects of ergotism.

Alkaloid Biosynthetic Enzyme Generates Diastereomeric Pair via Two Distinct Mechanisms.

Ergopeptines constitute one of the representative classes of ergoline alkaloids and carry a tripeptide extension on the lysergic acid core. In the current study, we discovered and structurally characterized newly isolated ergopeptine-like compounds named lentopeptins from a filamentous fungus Aspergillus lentulus, a close relative of A. fumigatus. Interestingly, in lentopeptins, the common lysergic acid moiety of ergopeptines is replaced by a cinnamic acid moiety at the N-terminus of the peptide segment. Moreover, lentopeptins lack the C-terminal proline residue necessary for the spontaneous cyclization of the peptide extension. Herein, we report the atypical lentopeptin biosynthetic pathway identified through targeted deletion of the len cluster biosynthetic genes predicted from the genome sequence. Further in vitro characterizations of the thiolation-terminal condensation-like (T-CT) didomain of the nonribosomal peptide synthetase LenA and its site-specific mutants revealed the mechanism of peptide release via diketopiperazine formation, an activity previously unreported for CT domains. Most intriguingly, in vitro assays of the cytochrome P450 LenC illuminated the unique mechanisms to generate two diastereomeric products. Lentopeptin A forms via a stereospecific hydroxylation, followed by a spontaneous bicyclic lactam core formation, while lentopeptin B is produced through an initial dehydrogenation, followed by a bicyclic lactam core formation and stereospecific hydration. Our results showcase how nature exploits common biosynthetic enzymes to forge new complex natural products effectively (213/250).

Reconstituting the complete biosynthesis of D-lysergic acid in yeast.

The ergot alkaloids are a class of natural products known for their pharmacologically privileged molecular structure that are used in the treatment of neurological ailments, such as Parkinsonism and dementia. Their synthesis via chemical and biological routes are therefore of industrial relevance, but suffer from several challenges. Current chemical synthesis methods involve long, multi-step reactions with harsh conditions and are not enantioselective; biological methods utilizing ergot fungi, produce an assortment of products that complicate product recovery, and are susceptible to strain degradation. Reconstituting the ergot alkaloid pathway in a strain strongly amenable for liquid fermentation, could potentially resolve these issues. In this work, we report the production of the main ergoline therapeutic precursor, D-lysergic acid, to a titre of 1.7 mg L-1 in a 1 L bioreactor. Our work demonstrates the proof-of-concept for the biological production of ergoline-derived compounds from sugar in an engineered yeast chassis.

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 µg/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 µg/kg).

Cleaving Ergot Alkaloids by Hydrazinolysis-A Promising Approach for a Sum Parameter Screening Method.

Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods-acidic esterification and hydrazinolysis-are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40‒60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.