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Publikation

Fu, N.; Becker, T.; Brandt, W.; Kunert, M.; Burse, A.; Boland, W.; Involvement of CYP347W1 in neurotoxin 3‐nitropropionic acid based chemical defense in mustard leaf beetle Phaedon cochleariae Insect Sci. 29, 453–466, (2022) DOI: 10.1111/1744-7917.12944

Chrysomelina beetles store 3-nitropropionic acid in form of a pre-toxin, isoxazolin-5-one glucoside conjugated ester, to protect themselves against predators. Here we identified a cytochrome P450 monooxygenase, CYP347W1, to be involved in the production of the 3-nitropropionic acid moiety of the isoxazolin-5-one glucoside ester. Knocking down CYP347W1 led to a significant depletion in the concentration of the isoxazolin-5-one glucoside ester and an increase in the concentration of the isoxazolin-5-one glucoside in the larval hemolymph. Enzyme assays with the heterologously expressed CYP347W1 showed free β-alanine was not the direct substrate. Homology modeling indicated that β-alanine-CoA ester can fit into CYP347W1’s active site. Furthermore, we proved that Phaedon cochleariae eggs are not able to de novo synthesize 3-NPA, although both isoxazolin-5-one glucoside and its 3-NPA conjugated ester are present in the eggs. These results provide direct evidence for the involvement of CYP347W1 in the biosynthesis of a P. cochleariae chemical defense compound.
Publikation

Vattekkatte, A.; Garms, S.; Brandt, W.; Boland, W.; Enhanced structural diversity in terpenoid biosynthesis: enzymes, substrates and cofactors Org. Biomol. Chem. 16, 348-362, (2018) DOI: 10.1039/C7OB02040F

The enormous diversity of terpenes found in nature is generated by enzymes known as terpene synthases, or cyclases. Some are also known for their ability to convert a single substrate into multiple products. This review comprises monoterpene and sesquiterpene synthases that are multiproduct in nature along with the regulation factors that can alter the product specificity of multiproduct terpene synthases without genetic mutations. Variations in specific assay conditions with focus on shifts in product specificity based on change in metal cofactors, assay pH and substrate geometry are described. Alterations in these simple cellular conditions provide the organism with enhanced chemodiversity without investing into new enzymatic architecture. This versatility to modulate product diversity grants organisms, especially immobile ones like plants with access to an enhanced defensive repertoire by simply altering cofactors, pH level and substrate geometry.
Publikation

Rahfeld, P.; Haeger, W.; Kirsch, R.; Pauls, G.; Becker, T.; Schulze, E.; Wielsch, N.; Wang, D.; Groth, M.; Brandt, W.; Boland, W.; Burse, A.; Glandular β-glucosidases in juvenile Chrysomelina leaf beetles support the evolution of a host-plant-dependent chemical defense Insect Biochem. Mol. Biol. 58, 28-38, (2015) DOI: 10.1016/j.ibmb.2015.01.003

Plant-feeding insects are spread across the entire plant kingdom. Because they chew externally on leaves, leaf beetle of the subtribe Chrysomelina sensu stricto are constantly exposed to life-threatening predators and parasitoids. To counter these pressures, the juveniles repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors. The autonomous production of iridoids pre-dates the evolution of phytochemical-based defense strategies. Both strategies include hydrolysis of the secreted non-toxic glycosides in the defensive exudates. By combining in vitro as well as in vivo experiments, we show that iridoid de novo producing as well as sequestering species rely on secreted β-glucosidases to cleave the pre-toxins. Our phylogenetic analyses support a common origin of chrysomeline β-glucosidases. The kinetic parameters of these β-glucosidases demonstrated substrate selectivity which reflects the adaptation of Chrysomelina sensu stricto to the chemistry of their hosts during the course of evolution. However, the functional studies also showed that the broad substrate selectivity allows building a chemical defense, which is dependent on the host plant, but does not lead to an “evolutionary dead end”.
Publikation

Vattekkatte, A.; Gatto, N.; Schulze, E.; Brandt, W.; Boland, W.; Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates Org. Biomol. Chem. 13, 4776-4784, (2015) DOI: 10.1039/c5ob00506j

The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.
Publikation

Nakamura, Y.; Paetz, C.; Brandt, W.; David, A.; Rendón-Anaya, M.; Herrera-Estrella, A.; Mithöfer, A.; Boland, W.; Synthesis of 6-Substituted 1-oxoindanoyl Isoleucine Conjugates and Modeling Studies with the COI1-JAZ Co-Receptor Complex of Lima Bean J. Chem. Ecol. 40, 687-699, (2014) DOI: 10.1007/s10886-014-0469-2

The conjugates of 6-substituted 1-oxoindanoyl carboxylic acids with L-isoleucine are mimics of the plant hormone (+)-7-iso-JA-L-Ile (3) that controls and regulates secondary metabolism and stress responses. In order to generate ligands that can be used as hormone-like compounds possessing different biological activities, an efficient and short synthesis of 6-bromo-1-oxoindane-4-carboxylic acid opens a general route to 6-Br-1-oxoindanoyl L-isoleucine conjugate (Br-In-L-Ile) (9a) as a key intermediate for several bioactive 6-halogen-In-L-Ile analogs (7a, 8a, 10a). The 6-ethynyl-In-L-Ile analog (11a) might be a valuable tool to localize macromolecular receptor molecules by click-chemistry. The activities of In-Ile derivatives were evaluated by assays inducing the release of volatile organic compounds (VOCs) in lima bean (Phaseolus lunatus). Each compound showed slightly different VOC induction patterns. To correlate such differences with structural features, modeling studies of In-Ile derivatives with COI-JAZa/b/c co-receptors of P. lunatus were performed. The modeling profits from the rigid backbone of the 1-oxoindanonoyl conjugates, which allows only well defined interactions with the receptor complex.
Publikation

Frick, S.; Nagel, R.; Schmidt, A.; Bodemann, R. R.; Rahfeld, P.; Pauls, G.; Brandt, W.; Gershenzon, J.; Boland, W.; Burse, A.; Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway Proc. Natl. Acad. Sci. U.S.A. 110, 4194-4199, (2013) DOI: 10.1073/pnas.1221489110

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.
Publikation

Jimenez-Aleman, G. H.; Schöner, T.; Montero-Alejo, A. L.; Brandt, W.; Boland, W.; Improved synthesis of the chrysomelid pheromone (6R,7S)-(+)-himachala-9,11-diene via spontaneous bromination and didehydrobromination of 2,6,6,9-tetramethyl-bicyclo[5.4.0]undec-8-ene ARKIVOC 2012, 371-378, (2012) DOI: 10.3998/ark.5550190.0013.326

A convenient synthesis of (6R,7S)-(+)-himachala-9,11-diene, the pheromone of the chrysomelid beetle Phyllotreta striolata is described. The diene is obtained in a single operation by a spontaneous “bromination/dehydrobromination” of 2,6,6,9-tetramethylbicyclo[5.4.0]undec-8-ene. The halogenation/dehalogenation sequence proceeds spontaneously in CCl4, and is less uniform in CH2Cl2 and CHCl3. 1H NMR experiments carried out in presence of the radical scavenger di-tert-butyl-4-methylphenol suggest an ionic mechanism for this reaction. Theoretical calculations demonstrate that the spontaneous reaction profits from the strongly exergonic addition of Br2 to the double bond and an almost neutral energy difference between the starting olefin and the diene pheromone.
Publikation

Vadassery, J.; Reichelt, M.; Hause, B.; Gershenzon, J.; Boland, W.; Mithöfer, A.; CML42-Mediated Calcium Signaling Coordinates Responses to Spodoptera Herbivory and Abiotic Stresses in Arabidopsis Plant Physiol. 159, 1159-1175, (2012) DOI: 10.1104/pp.112.198150

In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca2+ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca2+ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca2+ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca2+ sensor having multiple functions in insect herbivory defense and abiotic stress responses.
Publikation

Dąbrowska, P.; Shabab, M.; Brandt, W.; Vogel, H.; Boland, W.; Isomerization of the Phytohormone Precursor 12-Oxophytodienoic Acid (OPDA) in the Insect Gut J. Biol. Chem. 286, 22348-22354, (2011) DOI: 10.1074/jbc.M111.244509

12-Oxophytodienoic acid (OPDA) is isomerized in the gut of herbivorous insects to tetrahydrodicranenone B (iso-OPDA). The transformation is achieved by a glutathione S-transferase present in the gut epithelium. Experiments with 9-[2H]-iso-OPDA demonstrated the complete retention of the deuterium atom in the product 11-[2H]-OPDA consistent with an intramolecular 1,3-hydrogen shift. Homology modeling based on the x-ray structure of a glutathione S-transferase from Anopheles gambiae revealed that the co-factor glutathione does not covalently bind to the substrate but appears to be involved in the initial deprotonation and enolization of the OPDA. The transformation resembles that of a mammalian GST-catalyzed isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids or the conversion of prostaglandin A1 to the biologically inactive prostaglandin B1.
Publikation

Paetzold, H.; Garms, S.; Bartram, S.; Wieczorek, J.; Urós-Gracia, E.-M.; Rodríguez-Concepción, M.; Boland, W.; Strack, D.; Hause, B.; Walter, M. H.; The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes Mol. Plant 3, 904-916, (2010) DOI: 10.1093/mp/ssq032

Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SlDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SlDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SlDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SlDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of 13C to 12C natural isotope ratios. A compensatory up-regulation of SlDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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