DOCSLIB.ORG

Structure of an Aspergillus Fumigatus Old Yellow Communications Enzyme (Easa) Involved in Ergot Alkaloid ISSN 2053-230X Biosynthesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structure of an Aspergillus Fumigatus Old Yellow Communications Enzyme (Easa) Involved in Ergot Alkaloid ISSN 2053-230X Biosynthesis structural communications Acta Crystallographica Section F Structural Biology Structure of an Aspergillus fumigatus old yellow Communications enzyme (EasA) involved in ergot alkaloid ISSN 2053-230X biosynthesis Annemarie S. Chilton, Ashley L. The Aspergillus fumigatus old yellow enzyme (OYE) EasA reduces chano- Ellis and Audrey L. Lamb* clavine-I aldehyde to dihydrochanoclavine aldehyde and works in conjunction with festuclavine synthase at the branchpoint for ergot alkaloid pathways. The ˚ Molecular Biosciences, University of Kansas, crystal structure of the FMN-loaded EasA was determined to 1.8 A resolution. 1200 Sunnyside Avenue, Lawrence, KS 66045, The active-site amino acids of OYE are conserved, supporting a similar USA mechanism for reduction of the / -unsaturated aldehyde. The C-terminal tail of one monomer packs into the active site of a monomer in the next asymmetric unit, which is most likely to be a crystallization artifact and not a mechanism of Correspondence e-mail: [email protected] self-regulation. Received 22 July 2014 1. Introduction Accepted 21 August 2014 Ergot alkaloids are produced by fungi and plants and have histori- cally been used medicinally and for psychedelic recreation (lysergic acid derivatives), and have been the cause of poisonings with symp- PDB reference: EasA, 4qnw toms that were attributed to witchcraft (Schardl et al., 2006). These secondary metabolites are derived from tryptophan and dimethylallyl diphosphate (DMAPP) and are characterized by the tetracyclic ergoline ring system, of which festuclavine is one of the simplest examples, or characterized by the tricyclic ring system exemplified by chanoclavine-I aldehyde (Fig. 1a; Schardl et al., 2006). Isotope- feeding studies suggest that chanoclavine-I aldehyde may be a common intermediate for the biosynthesis of all ergot alkaloids (Floss et al., 1974). Conversion of chanoclavine-I aldehyde to dihy- drochanoclavine aldehyde by Aspergillus fumigatus has been reported to require an old yellow enzyme (OYE) called EasA (Cheng et al., 2010a,b; Coyle et al., 2010; Geu-Flores et al., 2012) or FgaOx3 (Wallwey et al., 2010) for the reduction of the / -unsaturated alde- hyde. The dihydrochanoclavine aldehyde is subsequently cyclized to form an iminium compound. It is unknown whether the cyclization is enzyme-catalyzed. A second reduction step to produce festuclavine from the iminium compound is performed by the NADH-dependent festuclavine synthase FgaFS (Wallwey et al., 2010). The Saccharomyces pastorianus old yellow enzyme was the first flavoenzyme to be identified (Warburg & Christian, 1932). Members of the OYE family are found in bacteria, fungi and plants, and catalyze the reduction of activated carbon–carbon double bonds (for excellent reviews, see Stuermer et al., 2007; Williams & Bruce, 2002). Here, we present the structural characterization of the A. fumigatus OYE EasA involved in ergot alkaloid biosynthesis. These data allow us to corroborate the proposed mechanism and to provide insight into the oligomerization of EasA in solution and in the crystal. 2. Materials and methods 2.1. Macromolecule production The preparation of the expression vector has been described previously (Cheng et al., 2010a). The expression vector, which generates C-terminally histidine-tagged EasA, was transformed into Escherichia coli BL21(DE3) cells, which were grown in LB broth containing 50 mgmlÀ1 kanamycin at 37C with shaking (175 rev minÀ1) for 2.5 h. The temperature was reduced to 15C for # 2014 International Union of Crystallography 1 h until an optical density at 600 nm of 0.1 was reached. Protein All rights reserved expression was induced with 50 mM IPTG and the cultures were 1328 doi:10.1107/S2053230X14018962 Acta Cryst. (2014). F70, 1328–1332 structural communications incubated for 64 h. Cells were harvested by centrifugation (3800g, (GE Healthcare) pre-equilibrated with 50 mM Tris–HCl, 150 mM 4C, 10 min). The cell pellet was resuspended in 10 ml 5 mM imida- sodium citrate, 10% glycerol pH 8.0. The fractions containing EasA zole, 300 mM NaCl, 20 mM Tris–HCl pH 8.0, 10% glycerol (buffer A). were pooled, concentrated to 3 mg mlÀ1 using an Amicon stirred cell The cells were disrupted using a French pressure cell (241 MPa) and with a YM-30 membrane and stored at À80C. Approximately 9 mg cellular debris was removed by centrifugation (20 800g,4C, 1 h). The EasA was purified from 4 l culture. EasA eluted from the size- lysate was applied onto 50 ml Ni–NTA resin (Qiagen) in a Kontes exclusion column was 85% FMN-bound. The amount of FMN column equilibrated with buffer A. The column was shaken to mix the loading was determined by comparing the concentration of EasA, lysate with the resin and incubated overnight at 4 C on a gently tilting determined by measuring the A280 (in deionized water " = base. After 21 h, EasA was eluted from the Ni–NTA column with a 56 840 MÀ1 cmÀ1), and the concentration of FMN, determined by À1 À1 step gradient of imidazole (5, 25, 50, 100, 150 and 300 mM) in buffer measuring the A446 (" = 11 500 M cm ). A. EasA primarily eluted at 150 mM imidazole. To load the EasA protein with FMN (flavin mononucleotide), a 2.2. Crystallization previously published procedure (Gatti et al., 1994) was modified. A Crystals were grown at 25C using the hanging-drop vapor-diffu- Red Sepharose (GE Healthcare) column was pre-equilibrated with sion method with 750 ml reservoir solution consisting of 230 mM three column volumes of buffer B (50 mM potassium phosphate ammonium sulfate, 100 mM MES pH 6.0, 30% PEG 8000. The monobasic pH 7.0, 10% glycerol). The 150 mM imidazole Ni–NTA hanging drops consisted of 1.5 ml reservoir solution and 1.5 ml fractions (50 ml) were concentrated to 25 ml and diluted in buffer B 24 mg mlÀ1 protein. Yellow crystals were fully developed in 4 d with to a final estimated salt concentration of 25 mM NaCl. The sample approximate dimensions of 160 Â 40 Â 20 mm. For data collection, was injected and washed with eight column volumes of buffer B crystals were transferred to the reservoir solution supplemented with followed by a linear gradient of 0–600 mM NaCl (five column 20% glycerol solution and were flash-cooled by plunging them into volumes) in buffer B to wash out unbound sample. The column was liquid nitrogen. washed with two column volumes of buffer B with 10 mM FMN. EasA was eluted using a 20-column-volume linear gradient from 0 to 2 M NaCl with 10 mM FMN in buffer B. 2.3. Data collection and processing The fractions containing EasA from the Red Sepharose column X-ray diffraction data were collected on beamline 11-1 at the were pooled and applied onto a Superdex 200 size-exclusion column Stanford Synchrotron Radiation Laboratory (SSRL), Stanford, Figure 1 EasA mechanism and overall structure. (a) The reaction catalyzed by the old yellow enzyme EasA from A. fumigatus, the conversion of chanoclavine-I aldehyde to dihydrochanoclavine, is labeled in red. The next two reactions are hypothesized to be spontaneous. The final reaction for the generation of festuclavine is catalyzed by FgaFS, festuclavine synthase. (b) EasA is a classic / -barrel, shown as a blue cartoon. The FMN (green sticks) is bound in the active-site funnel composed by eight parallel strands of the -barrel. The backside of the barrel is closed by a two-stranded -sheet: the two strands that precede the first strand of the barrel at the N-terminus of the protein. (c) The C-terminal tail of the red monomer packs in the active site of the blue monomer in the next asymmetric unit. The location of the active site is noted by the inclusion of the FMN as green sticks. Protein structure pictures were generated in PyMOL (Schro¨dinger). Acta Cryst. (2014). F70, 1328–1332 Chilton et al. EasA 1329 structural communications Table 1 Table 2 Data collection and processing. Structure refinement. Values in parentheses are for the outer shell. Values in parentheses are for the outer shell. Diffraction source SSRL beamline 11-1 Resolution range (A˚ ) 35.630–1.799 (1.846–1.799) Wavelength (A˚ ) 0.97945 No. of reflections 40082 (2851) Temperature ( C) À173 Final Rcryst 0.178 (0.221) Detector PILATUS 6M Final Rfree 0.221 (0.273) Crystal-to-detector distance (mm) 260 Total unique observations 40082 Rotation range per image ()0.5No. of non-H atoms Total rotation range () 180 Protein 2916 Exposure time per image (s) 20 Solvent (sulfate and glycerol) 26 Space group P212121 FMN 31 a, b, c (A˚ ) 62.5, 65.9, 115.2 Water 239 , , () 90, 90, 90 R.m.s. deviations Resolution range (A˚ ) 38.41–1.80 (1.90–1.80) Bonds (A˚ ) 0.026 Total No. of reflections 162475 (23165) Angles () 2.02 No. of unique reflections 44595 (6332) Overall mean B factor (A˚ 2) 21.6 Completeness (%) 99.3 (97.6) Multiplicity 3.6 (3.7) hI/(I)i 23.6 (3.3) percentage of FMN bound (0–6%) by a comparable purification R 0.040 (0.399) merge strategy using nickel-affinity chromatography (the protein is histi- dine-tagged). However, a purification step was added in which Red Califonia, USA using a wavelength of 0.97945 A˚ . Data (180) were Sepharose is used to bind EasA (owing to its affinity for the flavin) collected as 0.5 oscillation images (20 s exposure per frame) using and the protein is eluted from the column using sodium chloride in Blu-Ice (Soltis et al., 2008) with a crystal-to-detector distance of the presence of FMN. This is an adaptation of the protocol used to 260 mm at À173C. The crystals were assigned to space group exchange the natural flavin in p-hydroxybenzoate hydroxylase for 6- ˚ P212121, with unit-cell parameters a = 62.5, b = 65.9, c = 115.2 A.The azido FAD (Gatti et al., 1994).
Load more

Recommended publications
  • Ergot Alkaloid Biosynthesis in Aspergillus Fumigatus : Association with Sporulation and Clustered Genes Common Among Ergot Fungi
    Graduate Theses, Dissertations, and Problem Reports 2009 Ergot alkaloid biosynthesis in Aspergillus fumigatus : Association with sporulation and clustered genes common among ergot fungi Christine M. Coyle West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Coyle, Christine M., "Ergot alkaloid biosynthesis in Aspergillus fumigatus : Association with sporulation and clustered genes common among ergot fungi" (2009). Graduate Theses, Dissertations, and Problem Reports. 4453. https://researchrepository.wvu.edu/etd/4453 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Ergot alkaloid biosynthesis in Aspergillus fumigatus: Association with sporulation and clustered genes common among ergot fungi Christine M. Coyle Dissertation submitted to the Davis College of Agriculture, Forestry, and Consumer Sciences at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics and Developmental Biology Daniel G. Panaccione, Ph.D., Chair Kenneth P. Blemings, Ph.D. Joseph B.
    [Show full text]
  • Risk Assessment of Argyreia Nervosa
    Risk assessment of Argyreia nervosa RIVM letter report 2019-0210 W. Chen | L. de Wit-Bos Risk assessment of Argyreia nervosa RIVM letter report 2019-0210 W. Chen | L. de Wit-Bos RIVM letter report 2019-0210 Colophon © RIVM 2020 Parts of this publication may be reproduced, provided acknowledgement is given to the: National Institute for Public Health and the Environment, and the title and year of publication are cited. DOI 10.21945/RIVM-2019-0210 W. Chen (author), RIVM L. de Wit-Bos (author), RIVM Contact: Lianne de Wit Department of Food Safety (VVH) [email protected] This investigation was performed by order of NVWA, within the framework of 9.4.46 Published by: National Institute for Public Health and the Environment, RIVM P.O. Box1 | 3720 BA Bilthoven The Netherlands www.rivm.nl/en Page 2 of 42 RIVM letter report 2019-0210 Synopsis Risk assessment of Argyreia nervosa In the Netherlands, seeds from the plant Hawaiian Baby Woodrose (Argyreia nervosa) are being sold as a so-called ‘legal high’ in smart shops and by internet retailers. The use of these seeds is unsafe. They can cause hallucinogenic effects, nausea, vomiting, elevated heart rate, elevated blood pressure, (severe) fatigue and lethargy. These health effects can occur even when the seeds are consumed at the recommended dose. This is the conclusion of a risk assessment performed by RIVM. Hawaiian Baby Woodrose seeds are sold as raw seeds or in capsules. The raw seeds can be eaten as such, or after being crushed and dissolved in liquid (generally hot water).
    [Show full text]
  • Diversification of Ergot Alkaloids in Natural and Modified Fungi
    Toxins 2015, 7, 201-218; doi:10.3390/toxins7010201 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Review Diversification of Ergot Alkaloids in Natural and Modified Fungi Sarah L. Robinson and Daniel G. Panaccione * Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506, USA; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-304-293-8819; Fax: +1-304-293-2960. Academic Editor: Christopher L. Schardl Received: 21 November 2014 / Accepted: 14 January 2015 / Published: 20 January 2015 Abstract: Several fungi in two different families––the Clavicipitaceae and the Trichocomaceae––produce different profiles of ergot alkaloids, many of which are important in agriculture and medicine. All ergot alkaloid producers share early steps before their pathways diverge to produce different end products. EasA, an oxidoreductase of the old yellow enzyme class, has alternate activities in different fungi resulting in branching of the pathway. Enzymes beyond the branch point differ among lineages. In the Clavicipitaceae, diversity is generated by the presence or absence and activities of lysergyl peptide synthetases, which interact to make lysergic acid amides and ergopeptines. The range of ergopeptines in a fungus may be controlled by the presence of multiple peptide synthetases as well as by the specificity of individual peptide synthetase domains. In the Trichocomaceae, diversity is generated by the presence or absence of the prenyl transferase encoded by easL (also called fgaPT1). Moreover, relaxed specificity of EasL appears to contribute to ergot alkaloid diversification. The profile of ergot alkaloids observed within a fungus also is affected by a delayed flux of intermediates through the pathway, which results in an accumulation of intermediates or early pathway byproducts to concentrations comparable to that of the pathway end product.
    [Show full text]
  • Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases
    G C A T T A C G G C A T genes Review Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases Juan F. Martín *, Rubén Álvarez-Álvarez ID and Paloma Liras Department of Molecular Biology, Section of Microbiology, University of León, 24071 León, Spain; [email protected] (R.Á.-Á.); [email protected] (P.L.) * Correspondence: [email protected] Received: 19 October 2017; Accepted: 16 November 2017; Published: 24 November 2017 Abstract: The clavine alkaloids produced by the fungi of the Aspergillaceae and Arthrodermatacea families differ from the ergot alkaloids produced by Claviceps and Neotyphodium. The clavine alkaloids lack the extensive peptide chain modifications that occur in lysergic acid derived ergot alkaloids. Both clavine and ergot alkaloids arise from the condensation of tryptophan and dimethylallylpyrophosphate by the action of the dimethylallyltryptophan synthase. The first five steps of the biosynthetic pathway that convert tryptophan and dimethylallyl-pyrophosphate (DMA-PP) in chanoclavine-1-aldehyde are common to both clavine and ergot alkaloids. The biosynthesis of ergot alkaloids has been extensively studied and is not considered in this article. We focus this review on recent advances in the gene clusters for clavine alkaloids in the species of Penicillium, Aspergillus (Neosartorya), Arthroderma and Trychophyton and the enzymes encoded by them. The final products of the clavine alkaloids pathways derive from the tetracyclic ergoline ring, which is modified by late enzymes, including a reverse type prenyltransferase, P450 monooxygenases and acetyltransferases. In Aspergillus japonicus, a α-ketoglutarate and Fe2+-dependent dioxygenase is involved in the cyclization of a festuclavine-like unknown type intermediate into cycloclavine.
    [Show full text]
  • Ergot Alkaloid Biosynthesis
    Natural Product Reports Ergot alkaloid biosynthesis Journal: Natural Product Reports Manuscript ID: NP-HIG-05-2014-000062.R1 Article Type: Highlight Date Submitted by the Author: 03-Aug-2014 Complete List of Authors: Jakubczyk, Dorota; John Innes, Cheng, John; John Innes, O'Connor, Sarah; John Innes Centre, Norwich Research Park Page 1 of 14 Natural Product Reports Biosynthesis of the Ergot Alkaloids Dorota Jakubczyk, Johnathan Z. Cheng, Sarah E. O’Connor The John Innes Centre, Department of Biological Chemistry, Norwich NR4 7UH [email protected] 1. History of Ergot Alkaloids 2. Ergot Alkaloid Classes 3. Ergot Alkaloid Producers 4. Ergot Alkaloid Biosynthesis 4.1 Proposed Ergot Alkaloid Biosynthetic Pathway 4.2 Ergot Alkaloid Biosynthetic Gene Clusters 4.3 Functional Characterization of Early Ergot Alkaloid Biosynthetic Enzymes 4.4 Functional Characterization of Late Ergot Alkaloid Biosynthetic Enzymes 5. Production of Ergot Alkaloids 6. Conclusions The ergots are a structurally diverse group of alkaloids derived from tryptophan 7 and dimethylallyl pyrophosphate (DMAPP) 8. The potent bioactivity of ergot alkaloids have resulted in their use in many applications throughout human history. In this highlight, we recap some of the history of the ergot alkaloids, along with a brief description of the classifications of the different ergot structures and producing organisms. Finally we describe what the advancements that have been made in understanding the biosynthetic pathways, both at the genomic and the biochemical levels. We note that several excellent review on the ergot alkaloids, including one by Wallwey and Li in Nat. Prod. Rep., have been published recently.(1-3) We provide a brief overview of the ergot alkaloids, and highlight the advances in biosynthetic pathway elucidation that have been made since 2011 in section 4.
    [Show full text]
  • I ASPECTS of ERGOT ALKALOID BIOSYNTHESIS a Thesis Presented
    I ASPECTS OF ERGOT ALKALOID BIOSYNTHESIS A thesis presented by FRANCOISE QUIGLEY In part fulfilment of the requirements for the degree of Doctor of Philosophy, of the University of London Imperial College November 1973 Biochemistry Department ABSTRACT Methods of isolation and purification of ergot alkaloids were investigated with reference to the following chromatographic techniques: thin layer chromatography (T.L.C.), gas liquid chromatography (G.L.C.) and the use of neutral and ion-exchange chromatography. Sephadex SP C-25 ion-exchange resin, in appropriate ionic form, selectively absorbed ergot alkaloids from culture extracts and gave not only a separation of the basic and amphoteric fractions but also separated the peptide and clavine ergot alkaloids. G.L.C. analysis of Claviceps fusiformis extracts was achieved by trifluoroacetylation and silylation. The three major ergot alkaloids present in the amphoteric fraction of Claviceps fusiformis extracts were identified as 4-dimethylallyl- tryptophan, clavicipitic acid, and N-methyl-4-dimethylallyltryptophan. The latter, a new isolate, was observed to accumulate under anaerobic conditions. The inter-relationship of the clavine alkaloids was investigated in 14 Claviceps fusiformis by a kinetic feeding experiment using (2- C) tryptophan as the precursor. Results agreed with the generally accepted scheme of ergot alkaloid biosynthesis in Claviceps spp. tryptophan dimethylallyl- mevalonic ---4 tryptophan ___4 chanoclavine-I ---> agroclavine acid elymoclavine The presence of other biosynthetic intermediates, hydroxylated clavines, _ 14 was also suggested. Feeding of (3'-2H3, 2- C) mevalonate confirmed the general scheme above and provided further evidence for the loss from ) C-3 of mevalonate,of 1 and 2 protons in the enzymatic formation of chanoclavine I and agroclavine respectively.
    [Show full text]
  • Ergot Alkaloids Structure Diversity, Biosynthetic Gene Clusters And
    View Online NPR Dynamic Article LinksC< Cite this: Nat. Prod. Rep., 2011, 28, 496 www.rsc.org/npr REVIEW Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes Christiane Wallwey and Shu-Ming Li* Received 26th October 2010 DOI: 10.1039/c0np00060d Covering: 2000 to 2010 Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identified in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e. amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Significant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identified in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins. Downloaded by NUI Galway on 25 February 2011 1 Introduction 5.2 Identification of gene clusters from genome Published on 24 December 2010 http://pubs.rsc.org
    [Show full text]
  • 7. Ergot Alkaloids and Other Metabolites of the Genus Claviceps
    7. ERGOT ALKALOIDS AND OTHER METABOLITES OF THE GENUS CLAVICEPS MARTIN BUCHTA and LADISLAV CVAK Galena a.s. Opava, 747 70 Czech Republic 7.1. INTRODUCTION Both parasitic and saprophytic ergot is a producer of vast number of compounds from which alkaloids represent the most important group. Over 80 ergot alkaloids (EA) have as yet been isolated from diverse natural material, mainly from Claviceps strains, but also from other fungi and higher plants. Besides alkaloids and some other secondary metabolites (pigments and mycotoxins), ergot produces also some unusual primary metabolites of the lipidic and sacharidic nature. 7.2. ERGOT ALKALOIDS Only alkaloids isolated from different Claviceps species are described in this chapter. EA produced by other fungi and higher plants are the subject of the Chapter 18. Beside this limitation, nearly 70 ergot alkaloids, produced by genus Claviceps, are discussed here. 7.2.1. Ergot Alkaloid Structure All the EA are biosynthetically derived from L-tryptophane, that means indole is the base of their structure. Although the most of EA contains a tetra-cyclic ergoline system (Figure 1), there are some exceptions whose structure is created only by two or three cycles. Natural alkaloids can be divided into three groups according to their structure: The first, structurally simplest group, contains alkaloids of clavine type, the second group are simple amides of lysergic and paspalic acids and the third, most complex group is created by the alkaloids of peptidic type. Biosynthetically, the peptidic EA can be understand as tetrapeptides containing lysergic acid as the first member of the peptidic chain. The other amino acids of the tetrapeptide are variable, which provides the basis for a great diversity of this group of EA.
    [Show full text]
  • Recent Progress in Ergot Alkaloid Research†
    RSC Advances View Article Online REVIEW View Journal | View Issue Recent progress in ergot alkaloid research† Cite this: RSC Adv.,2017,7,27384 Jing-Jing Chen, Meng-Yao Han, Ting Gong, Jin-Ling Yang and Ping Zhu * Ergot alkaloids are a class of indole derivatives produced by the genera of Ascomycota including Claviceps, Aspergillus, Penicillium, and Epichloe¨. Many natural and semi-synthetic ergot alkaloids exhibit valuable pharmacological activities and have been widely used in the therapy of human CNS disorders. Owing to the development of genome sequencing technology, the gene clusters involved in the biosynthesis of ergot alkaloids have been identified from these fungi. In this review, we briefly introduce the pharmacological activities and possible mechanisms of action of some ergot alkaloids. Then we summarize the recent progress in the functional characterization of the key genes and gene clusters involved in the biosynthetic pathways of ergot alkaloids from different genera. Particularly, we Received 17th March 2017 summarize and discuss the constructions of ergot alkaloid biosynthetic pathways in different Accepted 17th May 2017 heterologous hosts and the optimization strategies performed on the recombinant strains, which provide DOI: 10.1039/c7ra03152a references for producing ergot alkaloids and the derivatives in cell factories by synthetic biology in the Creative Commons Attribution 3.0 Unported Licence. rsc.li/rsc-advances future. 1. Introduction cyclization of dimethylallyl pyrophosphate (DMAPP) and L- tryptophan. As shown in Fig. 1, based on their structures, ergot Ergot alkaloids are a class of indole derivatives having biological alkaloids are usually divided into three classes.1,9,10 Clavine-type and medicinal activities.
    [Show full text]
  • Ergot Alkaloids Contribute to Virulence in an Insect Model of Invasive Aspergillosis Received: 1 June 2017 Daniel G
    www.nature.com/scientificreports OPEN Ergot alkaloids contribute to virulence in an insect model of invasive aspergillosis Received: 1 June 2017 Daniel G. Panaccione & Stephanie L. Arnold Accepted: 20 July 2017 Neosartorya fumigata (Aspergillus fumigatus) is the most common cause of invasive aspergillosis, a Published: xx xx xxxx frequently fatal lung disease primarily afecting immunocompromised individuals. This opportunistic fungal pathogen produces several classes of specialised metabolites including products of a branch of the ergot alkaloid pathway called fumigaclavines. The biosynthesis of the N. fumigata ergot alkaloids and their relation to those produced by alternate pathway branches in fungi from the plant-inhabiting Clavicipitaceae have been well-characterised, but the potential role of these alkaloids in animal pathogenesis has not been studied extensively. We investigated the contribution of ergot alkaloids to virulence of N. fumigata by measuring mortality in the model insect Galleria mellonella. Larvae were injected with conidia (asexual spores) of two diferent wild-type strains of N. fumigata and three diferent ergot alkaloid mutants derived by previous gene knockouts and difering in ergot alkaloid profles. Elimination of all ergot alkaloids signifcantly reduced virulence of N. fumigata in G. mellonella (P < 0.0001). Mutants accumulating intermediates but not the pathway end product fumigaclavine C also were less virulent than the wild type (P < 0.0003). The data indicate that ergot alkaloids contribute to virulence of N. fumigata in this insect model and that fumigaclavine C is important for full virulence. Ergot alkaloids are specialised metabolites derived from prenylated tryptophan and produced by several fungi representing diferent phylogenetic lineages and occupying diferent ecological niches1–6.
    [Show full text]
  • Toxicity Studies of Chanoclavine in Mice
    toxins Article Toxicity Studies of Chanoclavine in Mice Sarah C. Finch 1,* , John S. Munday 2 , Jan M. Sprosen 1 and Sweta Bhattarai 1 1 AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton 3240, New Zealand; [email protected] (J.M.S.); [email protected] (S.B.) 2 Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand; [email protected] * Correspondence: sarah.fi[email protected] Received: 16 April 2019; Accepted: 30 April 2019; Published: 2 May 2019 Abstract: Epichloë endophytes have been used successfully in pastoral grasses providing protection against insect pests through the expression of secondary metabolites. This approach could be extended to other plant species, such as cereals, reducing reliance on pesticides. To be successful, the selected endophyte must express secondary metabolites that are active against cereal insect pests without any secondary metabolite, which is harmful to animals. Chanoclavine is of interest as it is commonly expressed by endophytes and has potential insecticidal activity. Investigation of possible mammalian toxicity is therefore required. An acute oral toxicity study showed the median lethal dose of chanoclavine to be >2000 mg/kg. This allows it to be classified as category 5 using the globally harmonized system of classification and labelling of chemicals, and category 6.1E using the New Zealand Hazardous Substances and New Organisms (HSNO) hazard classes, the lowest hazard class under both systems of classification. A three-week feeding study was also performed, which showed chanoclavine, at a dose rate of 123.9 mg/kg/day, initially reduced food consumption but was resolved by day seven.
    [Show full text]
  • Identification of a Critical Gene in the Dihydroergot Alkaloid Pathway
    Graduate Theses, Dissertations, and Problem Reports 2015 Identification of a critical gene in the dihydroergot alkaloid pathway Yulia Bilovol Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Bilovol, Yulia, "Identification of a critical gene in the dihydroergot alkaloid pathway" (2015). Graduate Theses, Dissertations, and Problem Reports. 5212. https://researchrepository.wvu.edu/etd/5212 This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Identification of a critical gene in the dihydroergot alkaloid pathway Yulia Bilovol Thesis submitted to the Davis College of Agriculture, Natural Resources, and Design at West Virginia University in partial fulfillment of the requirements for the degree of Master of Science in Applied and Environmental Microbiology Daniel G. Panaccione, Ph.D., Chair Gary K. Bissonnette, Ph.D Alan J. Sexstone, Ph.D Division of Plant and Soil Sciences Morgantown, West Virginia 2015 Keywords: Ergot alkaloids, Aspergillus, mycotoxins, fungi Copyright 2015 Yulia Bilovol Abstract Identification of a critical gene in the dihydroergot alkaloid pathway Yulia Bilovol Ergot alkaloids, bioactive compounds produced by some species of fungi, have had significant impacts on agriculture and medicine.
    [Show full text]