zur Suche springenzur Navigation springenzum Inhalt springen

Publikationen - Natur- und Wirkstoffchemie

Sortieren nach: Erscheinungsjahr Typ der Publikation

Zeige Ergebnisse 1 bis 10 von 13.

Publikation

Naumann, C.; Heisters, M.; Brandt, W.; Janitza, P.; Alfs, C.; Tang, N.; Toto Nienguesso, A.; Ziegler, J.; Imre, R.; Mechtler, K.; Dagdas, Y.; Hoehenwarter, W.; Sawers, G.; Quint, M.; Abel, S.; Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development Curr. Biol. 32, 2189-2205, (2022) DOI: 10.1016/j.cub.2022.04.005

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi sta-tus are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼2 μmM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.
Publikation

Milde, R.; Schnabel, A.; Ditfe, T.; Hoehenwarter, W.; Proksch, C.; Westermann, B.; Vogt, T.; Chemical synthesis of trans 8-methyl-6-nonenoyl-CoA and functional expression unravel capsaicin synthase activity encoded by the Pun1 Locus Molecules 27, 6878, (2022) DOI: 10.3390/molecules27206878

Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, the genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes an amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched-chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate the enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in Escherichia coli, and provide information on its substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and highly specific for vanilloylamine. Partly purified from E. coli, the recombinant active enzyme is a monomeric protein of 51 kDa that is independent of additional co-factors or associated proteins, as previously proposed. These data can now be used to design capsaicin synthase variants with different properties and alternative substrate preferences.
Publikation

Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.; A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases Commun. Biol. 4, 562, (2021) DOI: 10.1038/s42003-021-02076-3

AbstractFungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.
Preprints

Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.; A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases bioRxiv (2020) DOI: 10.1101/2020.07.22.216432

Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.
Publikation

Tabassum, N.; Eschen-Lippold, L.; Athmer, B.; Baruah, M.; Brode, M.; Maldonado-Bonilla, L. D.; Hoehenwarter, W.; Hause, G.; Scheel, D.; Lee, J.; Phosphorylation‐dependent control of an RNA granule‐localized protein that fine‐tunes defence gene expression at a post‐transcriptional level Plant J. 101, 1023-1039, (2020) DOI: 10.1111/tpj.14573

Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).
Publikation

Smolikova, G.; Gorbach, D.; Lukasheva, E.; Mavropolo-Stolyarenko, G.; Bilova, T.; Soboleva, A.; Tsarev, A.; Romanovskaya, E.; Podolskaya, E.; Zhukov, V. A.; Tikhonovich, I.; Medvedev, S.; Hoehenwarter, W.; Frolov, A.; Bringing new methods to the seed proteomics platform: Challenges and perspectives Int. J. Mol. Sci. 21, 9162, (2020) DOI: 10.3390/ijms21239162

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.
Publikation

Soboleva, A.; Mavropulo-Stolyarenko, G.; Karonova, T.; Thieme, D.; Hoehenwarter, W.; Ihling, C.; Stefanov, V.; Grishina, T.; Frolov, A.; Multiple Glycation Sites in Blood Plasma Proteins as an Integrated Biomarker of Type 2 Diabetes Mellitus Int. J. Mol. Sci. 20, 2329, (2019) DOI: 10.3390/ijms20092329

Type 2 diabetes mellitus (T2DM) is one of the most widely spread metabolic diseases. Because of its asymptomatic onset and slow development, early diagnosis and adequate glycaemic control are the prerequisites for successful T2DM therapy. In this context, individual amino acid residues might be sensitive indicators of alterations in blood glycation levels. Moreover, due to a large variation in the half-life times of plasma proteins, a generalized biomarker, based on multiple glycation sites, might provide comprehensive control of the glycemic status across any desired time span. Therefore, here, we address the patterns of glycation sites in highly-abundant blood plasma proteins of T2DM patients and corresponding age- and gender-matched controls by comprehensive liquid chromatography-mass spectrometry (LC-MS). The analysis revealed 42 lysyl residues, significantly upregulated under hyperglycemic conditions. Thereby, for 32 glycation sites, biomarker behavior was demonstrated here for the first time. The differentially glycated lysines represented nine plasma proteins with half-lives from 2 to 21 days, giving access to an integrated biomarker based on multiple protein-specific Amadori peptides. The validation of this biomarker relied on linear discriminant analysis (LDA) with random sub-sampling of the training set and leave-one-out cross-validation (LOOCV), which resulted in an accuracy, specificity, and sensitivity of 92%, 100%, and 85%, respectively.
Preprints

Mamontova, T.; Lukasheva, E.; Mavropolo-Stolyarenko, G.; Proksch, C.; Bilova, T.; Kim, A.; Babakov, V.; Grishina, T.; Hoehenwarter, W.; Medvedev, S.; Smolikova, G.; Frolov, A.; Proteome Map of Pea (Pisum Sativum L.) Embryos Containing Different Amounts of Residual Chlorophylls Preprints (2018) DOI: 10.20944/preprints201812.0069.v1

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major economically important legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Obviously, changes in seed protein patterns might directly affect both of these aspects. Thus, here we address the pea seed proteome in more detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. Accordingly, 1938 and 1989 non-redundant proteins were identified in yellow and green pea seeds, in total. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP) potentially indicating the high efficiency of our experimental workflow. In total 981 protein groups could be assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.
Publikation

Mamontova, T.; Lukasheva, E.; Mavropolo-Stolyarenko, G.; Proksch, C.; Bilova, T.; Kim, A.; Babakov, V.; Grishina, T.; Hoehenwarter, W.; Medvedev, S.; Smolikova, G.; Frolov, A.; Proteome Map of Pea (Pisum sativum L.) Embryos Containing Different Amounts of Residual Chlorophylls Int. J. Mol. Sci. 19, 4066, (2018) DOI: 10.3390/ijms19124066

Due to low culturing costs and high seed protein contents, legumes represent the main global source of food protein. Pea (Pisum sativum L.) is one of the major legume crops, impacting both animal feed and human nutrition. Therefore, the quality of pea seeds needs to be ensured in the context of sustainable crop production and nutritional efficiency. Apparently, changes in seed protein patterns might directly affect both of these aspects. Thus, here, we address the pea seed proteome in detail and provide, to the best of our knowledge, the most comprehensive annotation of the functions and intracellular localization of pea seed proteins. To address possible intercultivar differences, we compared seed proteomes of yellow- and green-seeded pea cultivars in a comprehensive case study. The analysis revealed totally 1938 and 1989 nonredundant proteins, respectively. Only 35 and 44 proteins, respectively, could be additionally identified after protamine sulfate precipitation (PSP), potentially indicating the high efficiency of our experimental workflow. Totally 981 protein groups were assigned to 34 functional classes, which were to a large extent differentially represented in yellow and green seeds. Closer analysis of these differences by processing of the data in KEGG and String databases revealed their possible relation to a higher metabolic status and reduced longevity of green seeds.
Publikation

Frolov, A.; Didio, A.; Ihling, C.; Chantzeva, V.; Grishina, T.; Hoehenwarter, W.; Sinz, A.; Smolikova, G.; Bilova, T.; Medvedev, S.; The effect of simulated microgravity on the Brassica napus seedling proteome Funct. Plant Biol. 45, 440-452, (2018) DOI: 10.1071/FP16378

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
IPB Mainnav Search