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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

Loesche, A., Wiese, J., Sommerwerk, S., Simon, V., Brandt, W. & Csuk, R. Repurposing N,N '-bis-(arylamidino)-1,4-piperazinedicarboxamidines: An unexpected class of potent inhibitors of cholinesterases. Eur J Med Chem 125, 430-434, (2017) DOI: 10.1016/j.ejmech.2016.09.051

Drug repurposing (=drug repositioning) is an effective way to cut costs for the development of new therapeutics and to reduce the time-to-market time-span. Following this concept a small library of compounds was screened for their ability to act as inhibitors of acetyl- and butyrylcholinesterase. Picloxydine, an established antiseptic, was shown to be an inhibitor for both enzymes. Systematic variation of the aryl substituents led to analogs possessing almost the same good properties as gold standard galantamine hydrobromide.
Publikation

Cotrim, C. A., Weidner, A., Strehmel, N., Bisol, T. B., Meyer, D., Brandt, W., Wessjohann, L. A. & Stubbs, M. T. A distinct aromatic prenyltransferase associated with the futalosine pathway. ChemistrySelect 2, 9319-9325, (2017) DOI: 10.1002/slct.201702151

Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4-hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4-dihydroxy-2-naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so-called futalosine pathway, has been described in certain micro-organisms. Until now, five soluble enzymes (MqnA-MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp-rich motif that serves to distinguish between members of the UbiA superfamily.
Publikation

Wiemann, J., Karasch, J., Loesche, A., Heller, L., Brandt, W. & Csuk, R. Piperlongumine B and analogs are promising and selective inhibitors for acetylcholinesterase. Eur J Med Chem 139, 222-231, (2017) DOI: 10.1016/j.ejmech.2017.07.081

Piperlongumine B (19), an alkaloid previously isolated from long pepper (Piper longum) has been synthesized for the first time in a short sequence and in good yield together with 19 analogs. Screening of these compounds in Ellman's assays showed several of them to be good inhibitors of acetylcholinesterase while being less active for butyrylcholinesterase. Activity of the compounds increased with the ring size of the heterocycle, and a maximum of activity was observed for an analog holding 12 methylene groups in the aliphatic side chain. These compounds may be regarded as promising candidates for the development of efficient inhibitors of acetylcholinesterase being useful for the treatment of Alzheimer's disease
Publikation

Dieckow, J., Brandt, W., Hattermann, K., Schob, S., Schulze, U., Mentlein, R., Ackermann, P., Sel, S. & Paulsen, F. P. CXCR4 and CXCR7 Mediate TFF3-Induced Cell Migration Independently From the ERK1/2 Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 57, 56-65, (2016) DOI: 10.1167/iovs.15-18129

Purpose: Trefoil factor family (TFF) peptides, and in particular TFF3, are characteristic secretory products of mucous epithelia that promote antiapoptosis, epithelial migration, restitution, and wound healing. For a long time, a receptor for TFF3 had not yet been identified. However, the chemokine receptor CXCR4 has been described as a low affinity receptor for TFF2. Additionally, CXCR7, which is able to heterodimerize with CXCR4, has also been discussed as a potential TFF2 receptor. Since there are distinct structural similarities between the three known TFF peptides, this study evaluated whether CXCR4 and CXCR7 may also act as putative TFF3 receptors.

Methods: We evaluated the expression of both CXCR4 and CXCR7 in samples of human ocular surface tissues and cell lines, using RT-PCR, immunohistochemistry, and Western blot analysis. Furthermore, we studied possible binding interactions between TFF3 and the receptor proteins in an x-ray structure-based modeling system. Functional studies of TFF3–CXCR4/CXCR7 interaction were accomplished by cell culture–based migration assays, flow cytometry, and evaluation of activation of the mitogen-activated protein (MAP) kinase signaling cascade.

Results: We detected both receptors at mRNA and protein level in all analyzed ocular surface tissues, and in lesser amount in ocular surface cell lines. X-ray structure-based modeling revealed CXCR4 and CXCR7 dimers as possible binding partners to TFF3. Cell culture–based assays revealed enhanced cell migration under TFF3 stimulation in a conjunctival epithelial cell line, which was completely suppressed by blocking CXCR4 and/or CXCR7. Flow cytometry showed increased proliferation rates after TFF3 treatment, while blocking both receptors had no effect on this increase. Trefoil factor family 3 also activated the MAP kinase signaling cascade independently from receptor activity. 

Publikation

Weigl, S., Brandt, W., Langhammer, R. & Roos, W. The vacuolar proton-cation exchanger EcNHX1 generates pH-signals for the expression of secondary metabolism in Eschscholzia californica. Plant Physiol. 170, 1135-1148, (2016) DOI: 10.1104/pp.15.01570

Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade towards the expression of biosynthetic enzymes includes a) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine and b) a subsequent, transient efflux of vacuolar protons resulting in a peak of cytosolic H+. The present study demonstrates that one of the Na+/H+ antiporters acting at the tonoplast of Eschscholzia cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from cDNA (EcNHX1-4). RNAi-based, simultaneous silencing of EcNHX1, 3 and 4 resulted in stable cell lines with largely diminished capacities of a) sodium dependent efflux of vacuolar protons and b) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of Eschscholzia reconstituted the lack of Na+ dependent H+ efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na+ - dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H+. This finding was supported by 3D protein homology models which predict a plausible recognition site for LPC only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expresssion of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process. 

Publikation

Piechulla, B., Bartelt, R., Brosemann, A., Effmert, U., Bouwmeester, H., Hippauf, F. & Brandt, W. The α-terpineol to 1,8-cineole cyclization reaction of tobacco terpene synthases Plant Physiol. 172, 2120-2131, (2016) DOI: 10.1104/pp.16.01378

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or α-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of α-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of α-terpineol by a nucleophilic attack of water. During this attack, the α-terpinyl cation is stabilized by π-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of α-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(−)-α-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed. 

Publikation

Kaluđerović, G. N., Hernández-Corroto, E., Brandt, W., Zmejkovski, B. B. & Gómez-Ruiz, S. Palladium(II) complexes with R2edda-derived ligands. J Coord Chem 69, 1337-1345, (2016) DOI: 10.1080/00958972.2016.1168519

Four palladium(II) complexes with R2edda ligands, dichlorido(O,O′-dialkylethylenediamine-N,N′-diacetate)palladium(II) monohydrates, [PdCl2(R2edda)]∙H2O, R = Me, Et, n-Pr, i-Bu, and the new ligand precursor i-Bu2edda∙2HCl∙H2O, O,O′-diisobutylethylenediamine-N,N′-diacetate dihydrochloride monohydrate, were synthesized and characterized by IR, 1H and 13C NMR spectroscopy, and elemental analysis. DFT calculations were performed for the palladium(II) complexes and a high possibility for isomer formation due to stereogenic N ligand atoms was confirmed. Moreover, DFT simulations revealed energetic profile of isomer formation. Computational outcomes are in agreement with spectroscopic instrumental findings, both strongly indicating a non-stereoselective reaction between selected esters and K2[PdCl4], forming isomers.

Publikation

Faden, F., Ramezani, T., Mielke, S., Almudi, I., Nairz, K., Froehlich, M. S., Höckendorff, J., Brandt, W., Hoehenwarter, W., Dohmen, R. J., Schnittger, A. & Dissmeyer, N. Phenotypes on demand via switchable target protein degradation in multicellular organisms Nat Commun. 7, 12202, (2016) DOI: 10.1038/ncomms12202

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.

Publikation

Domik, D., Thürmer, A., Weise, T., Brandt, W., Daniel, R. & Piechulla, B. A Terpene Synthase is involved in the synthesis of the volatile organic compound sodorifen of Serratia plymuthica 4Rx13. Front. Microbiol. 7, 737, (2016) DOI: 10.3389/fmicb.2016.00737

Bacteria release a plethora of volatile organic compounds, including compounds with extraordinary structures. Sodorifen (IUPAC name: 1,2,4,5,6,7,8-heptamethyl-3-methylenebicyclo[3.2.1]oct-6-ene) is a recently identified and unusual volatile hydrocarbon that is emitted by the rhizobacterium Serratia plymuthica 4R×13. Sodorifen comprises a bicyclic ring structure solely consisting of carbon and hydrogen atoms, where every carbon atom of the skeleton is substituted with either a methyl or a methylene group. This unusual feature of sodorifen made a prediction of its biosynthetic origin very difficult and so far its biosynthesis is unknown. To unravel the biosynthetic pathway we performed genome and transcriptome analyses to identify candidate genes. One knockout mutant (SOD_c20750) showed the desired negative sodorifen phenotype. Here it was shown for the first time that this gene is indispensable for the synthesis of sodorifen and strongly supports the hypothesis that sodorifen descends from the terpene metabolism. SOD_c20750 is the first bacterial terpene cyclase isolated from Serratia spp. and Enterobacteriales. Homology modeling revealed a 3D structure, which exhibits a functional role of amino acids for intermediate cation stabilization (W325) and putative proton acception (Y332). Moreover, the size and hydrophobicity of the active site strongly indicates that indeed the enzyme may catalyze the unusual compound sodorifen.

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