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Publikation

Wils, C. R.; Brandt, W.; Manke, K.; Vogt, T.; A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase FEBS Lett. 587, 683-689, (2013) DOI: 10.1016/j.febslet.2013.01.040

Caffeoyl‐coenzyme A O‐methyltransferase (CCoAOMT)‐like proteins from plants display a conserved position specificity towards the meta‐position of aromatic vicinal dihydroxy groups, consistent with the methylation pattern observed in vivo. A CCoAOMT‐like enzyme identified from Arabidopsis thaliana encoded by the gene At4g26220 shows a strong preference for methylating the para position of flavanones and dihydroflavonols, whereas flavones and flavonols are methylated in the meta‐position. Sequence alignments and homology modelling identified several unique amino acids compared to motifs of other CCoAOMT‐like enzymes. Mutation of a single glycine, G46 towards a tyrosine was sufficient for a reversal of the unusual para‐ back to meta‐O‐methylation of flavanones and dihydroflavonols.
Publikation

Wessjohann, L.; Vogt, T.; Kufka, J.; Klein, R.; Prenyl- und Methyltransferasen in Natur und Synthese BIOspektrum 18, 22-25, (2012) DOI: 10.1007/s12268-012-0137-4

Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.
Publikation

Fellenberg, C.; van Ohlen, M.; Handrick, V.; Vogt, T.; The role of CCoAOMT1 and COMT1 in Arabidopsis anthers Planta 236, 51-61, (2012) DOI: 10.1007/s00425-011-1586-6

Arabidopsis caffeoyl coenzyme A dependent O-methyltransferase 1 (CCoAOMT1) and caffeic acid O-methyltransferase 1 (COMT1) display a similar substrate profile although with distinct substrate preferences and are considered the key methyltransferases (OMTs) in the biosynthesis of lignin monomers, coniferyl and sinapoylalcohol. Whereas CCoAOMT1 displays a strong preference for caffeoyl coenzyme A, COMT1 preferentially methylates 5-hydroxyferuloyl CoA derivatives and also performs methylation of flavonols with vicinal aromatic dihydroxy groups, such as quercetin. Based on different knockout lines, phenolic profiling, and immunohistochemistry, we present evidence that both enzymes fulfil distinct, yet different tasks in Arabidopsis anthers. CCoAOMT1 besides its role in vascular tissues can be localized to the tapetum of young stamens, contributing to the biosynthesis of spermidine phenylpropanoid conjugates. COMT1, although present in the same organ, is not localized in the tapetum, but in two directly adjacent cells layers, the endothecium and the epidermal layer of stamens. In vivo localization and phenolic profiling of comt1 plants provide evidence that COMT1 neither contributes to the accumulation of spermidine phenylpropanoid conjugates nor to the flavonol glycoside pattern of pollen grains.
Publikation

Fellenberg, C.; Ziegler, J.; Handrick, V.; Vogt, T.; Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamens Front. Plant Sci. 3, 180, (2012) DOI: 10.3389/fpls.2012.00180

Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers.
Publikation

Wirsing, L.; Naumann, K.; Vogt, T.; Arabidopsis methyltransferase fingerprints by affinity-based protein profiling Anal. Biochem. 408, 220-225, (2011) DOI: 10.1016/j.ab.2010.09.029

Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR.
Publikation

Handrick, V.; Vogt, T.; Frolov, A.; Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry Anal. Bioanal. Chem. 398, 2789-2801, (2010) DOI: 10.1007/s00216-010-4129-2

Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.
Publikation

Vogt, T.; Phenylpropanoid Biosynthesis Mol. Plant 3, 2-20, (2010) DOI: 10.1093/mp/ssp106

The general phenylpropanoid metabolism generates an enormous array of secondary metabolites based on the few intermediates of the shikimate pathway as the core unit. The resulting hydroxycinnamic acids and esters are amplified in several cascades by a combination of reductases, oxygenases, and transferases to result in an organ and developmentally specific pattern of metabolites, characteristic for each plant species. During the last decade, methodology driven targeted and non-targeted approaches in several plant species have enabled the identification of the participating enzymes of this complex biosynthetic machinery, and revealed numerous genes, enzymes, and metabolites essential for regulation and compartmentation. Considerable success in structural and computational biology, combined with the analytical sensitivity to detect even trace compounds and smallest changes in the metabolite, transcript, or enzyme pattern, has facilitated progress towards a comprehensive view of the plant response to its biotic and abiotic environment. Transgenic approaches have been used to reveal insights into an apparently redundant gene and enzyme pattern required for functional integrity and plasticity of the various phenylpropanoid biosynthetic pathways. Nevertheless, the function and impact of all members of a gene family remain to be completely established. This review aims to give an update on the various facets of the general phenylpropanoid pathway, which is not only restricted to common lignin or flavonoid biosynthesis, but feeds into a variety of other aromatic metabolites like coumarins, phenolic volatiles, or hydrolyzable tannins.
Publikation

Lukačin, R.; Matern, U.; Hehmann, M.; Specker, S.; Vogt, T.; Corrigendum to “Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus” [FEBS Lett. 577 (2004) 367-370] FEBS Lett. 583, 855-855, (2009) DOI: 10.1016/j.febslet.2009.01.050

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Publikation

Fellenberg, C.; Böttcher, C.; Vogt, T.; Phenylpropanoid polyamine conjugate biosynthesis in Arabidopsis thaliana flower buds Phytochemistry 70, 1392-1400, (2009) DOI: 10.1016/j.phytochem.2009.08.010

Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.
Publikation

Kopycki, J. G.; Stubbs, M. T.; Brandt, W.; Hagemann, M.; Porzel, A.; Schmidt, J.; Schliemann, W.; Zenk, M. H.; Vogt, T.; Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 J. Biol. Chem. 283, 20888-20896, (2008) DOI: 10.1074/jbc.M801943200

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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