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Publikation

Brandt, W.; Schulze, E.; Liberman-Aloni, R.; Bartelt, R.; Pienkny, S.; Carmeli-Weissberg, M.; Frydman, A.; Eyal, Y.; Structural modeling of two plant UDP-dependent sugar-sugar glycosyltransferases reveals a conserved glutamic acid residue that is a hallmark for sugar acceptor recognition Journal of Structural Biology 213, 107777, (2021) DOI: 10.1016/j.jsb.2021.107777

Glycosylation is one of the common modifications of plant metabolites, playing a major role in the chemical/biological diversity of a wide range of compounds. Plant metabolite glycosylation is catalyzed almost exclusively by glycosyltransferases, mainly by Uridine-diphosphate dependent Glycosyltransferases (UGTs). Several X-ray structures have been determined for primary glycosyltransferases, however, little is known regarding structure–function aspects of sugar-sugar/branch-forming O-linked UGTs (SBGTs) that catalyze the transfer of a sugar from the UDP-sugar donor to an acceptor sugar moiety of a previously glycosylated metabolite substrate.In this study we developed novel insights into the structural basis for SBGT catalytic activity by modelling the 3d-structures of two enzymes; a rhamnosyl-transferase Cs1,6RhaT – that catalyzes rhamnosylation of flavonoid-3-glucosides and flavonoid-7-glucosides and a UGT94D1 – that catalyzes glucosylation of (+)-Sesaminol 2-O-β-Dglucoside at the C6 of the primary sugar moiety.Based on these structural models and docking studies a glutamate (E290 or E268 in Cs1,6RhaT or UGT94D1, respectively) and a tryptophan (W28 or W15 in Cs1,6RhaT or UGT94D1, respectively) appear to interact with the sugar acceptor and are suggested to be important for the recognition of the sugar-moiety of the acceptorsubstrate.Functional analysis of substitution mutants for the glutamate and tryptophan residues in Cs1,6RhaT further support their role in determining sugar-sugar/branch-forming GT specificity.Phylogenetic analysis of the UGT family in plants demonstrates that the glutamic-acid residue is a hallmark of SBGTs that is entirely absent from the corresponding position in primary UGTs.
Preprints

Zabel, S.; Brandt, W.; Porzel, A.; Athmer, B.; Kortbeek, R. W. J.; Bleeker, P. M.; Tissier, A.; Two novel 7-epi-zingiberene derivatives with biological activity from Solanum habrochaites are produced by a single cytochrome P450 monooxygenase bioRxiv (2020) DOI: 10.1101/2020.04.21.052571

Secretions from glandular trichomes potentially protect the plant against a variety of aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display substantial growth inhibitory activities against a range of microorganisms, including Bacillus subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.
Publikation

Jouda, J.-B.; Njoya, E. M.; Fobofou, S. A. T.; Zhou, Z. Y.; Qiang, Z.; Mbazoa, C. D.; Brandt, W.; Zhang, G.-l.; Wandji, J.; Wang, F.; Natural Polyketides Isolated from the Endophytic Fungus Phomopsis sp. CAM212 with a Semisynthetic Derivative Downregulating the ERK/IκBα Signaling Pathways Planta Med. 86, 1032-1042, (2020) DOI: 10.1055/a-1212-2930

AbstractThree previously undescribed natural products, phomopsinin A – C (1 – 3), together with three known compounds, namely, cis-hydroxymellein (4), phomoxanthone A (5) and cytochalasin L-696,474 (6), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1 and 2 were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1 yielded 1a, a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1 slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1, compound 1a, significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1a. The study of the mechanism of action revealed that 1a decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1β expression. Moreover, it was found that 1a exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1), such as compound 1a, could provide new anti-inflammatory leads.
Publikation

Holzmeyer, L.; Hartig, A.-K.; Franke, K.; Brandt, W.; Muellner-Riehl, A. N.; Wessjohann, L. A.; Schnitzler, J.; Evaluation of plant sources for antiinfective lead compound discovery by correlating phylogenetic, spatial, and bioactivity data Proc. Natl. Acad. Sci. U.S.A. 117, 12444-12451, (2020) DOI: 10.1073/pnas.1915277117

The continued high rates of using antibiotics in healthcare and livestock, without sufficient new compounds reaching the market, has led to a dramatic increase in antimicrobial resistance, with multidrug-resistant bacteria emerging as a major public health threat worldwide. Because the vast majority of antiinfectives are natural products or have originated from them, we assessed the predictive power of plant molecular phylogenies and species distribution modeling in the search for clades and areas which promise to provide a higher probability of delivering new antiinfective compound leads. Our approach enables taxonomically and spatially targeted bioprospecting and supports the battle against the global antimicrobial crisis.
Publikation

Fellenberg, C.; Corea, O.; Yan, L.; Archinuk, F.; Piirtola, E.; Gordon, H.; Reichelt, M.; Brandt, W.; Wulff, J.; Ehlting, J.; Constabel, C. P.; Discovery of salicyl benzoate UDP‐glycosyltransferase, a central enzyme in poplar salicinoid phenolic glycoside biosynthesis Plant J. 102, 99-115, (2020) DOI: 10.1111/tpj.14615

The salicinoids are anti‐herbivore phenolic glycosides unique to the Salicaceae (Populus and Salix). They consist of a salicyl alcohol glucoside core, which is usually further acylated with benzoic, cinnamic, or phenolic acids. While salicinoid structures are well known, their biosynthesis remains enigmatic. Recently, two enzymes from poplar, salicyl alcohol benzoyl transferase and benzyl alcohol benzoyl transferase, were shown to catalyze the production of salicyl benzoate, a predicted potential intermediate in salicinoid biosynthesis. Here, we used transcriptomics and co‐expression analysis with these two genes to identify two UDP‐glucose dependent glycosyltransferases (UGT71L1 and UGT78M1) as candidate enzymes in this pathway. Both recombinant enzymes accepted only salicyl benzoate, salicyl aldehyde, and 2‐hydroxycinnamic acid as glucose acceptors. Knocking out the UGT71L1 gene by CRISPR/Cas9 in poplar hairy root cultures led to the complete loss of salicortin, tremulacin, and tremuloidin, and a partial reduction of salicin content. This demonstrated that UGT71L1 is required for synthesis of the major salicinoids, and suggested that an additional route can lead to salicin. CRISPR/Cas9 knockouts for UGT78M1 were not successful, and its in vivo role thus remains to be determined. Although it has a similar substrate preference and predicted structure as UGT71L1, it appears not to contribute to the synthesis of salicortin, tremulacin, and tremuloidin, at least in roots. The demonstration of UGT71L1 as an enzyme of salicinoid biosynthesis will open up new avenues for the elucidation of this pathway.
Publikation

Böhme, B.; Moritz, B.; Wendler, J.; Hertel, T. C.; Ihling, C.; Brandt, W.; Pietzsch, M.; Enzymatic activity and thermoresistance of improved microbial transglutaminase variants Amino Acids 52, 313-326, (2020) DOI: 10.1007/s00726-019-02764-9

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein β-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.
Publikation

Vasco, A. V.; Moya, C. G.; Gröger, S.; Brandt, W.; Balbach, J.; Pérez, C. S.; Wessjohann, L. A.; Rivera, D. G.; Insights into the secondary structures of lactam N-substituted stapled peptides Org. Biomol. Chem. 18, 3838-3842, (2020) DOI: 10.1039/D0OB00767F

Stapled peptides derived from the Ugi macrocyclization comprise a special class of cyclopeptides with an N-substituted lactam bridge cross-linking two amino acid side chains. Herein we report a comprehensive analysis of the structural factors influencing the secondary structure of these cyclic peptides in solution. Novel insights into the s-cis/s-trans isomerism and the effect of N-functionalization on the conformation are revealed.
Publikation

Neubauer, P. R.; Pienkny, S.; Wessjohann, L. A.; Wessjohann, L.; Brandt, W.; Sewald, N.; Predicting the substrate scope of the flavin‐dependent halogenase BrvH ChemBioChem 21, 3282–3288, (2020) DOI: 10.1002/cbic.202000444

The recently described flavin‐dependent halogenase BrvH is able to catalyze both bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in vitro was docked into the active site of a complete protein model based on the X‐ray structure of BrvH. The atoms not resolved experimentally have been modelled using molecular mechanics force fields to obtain this protein model. Furthermore, docking poses for the substrates and known non‐substrates have been calculated. Parameters like distance, partial charge, and hybridization state have been analysed to derive rules for prediction of activity. With this model for activity of the BrvH, a virtual screening suggested several structures for potential substrates. Some of the thus preselected compounds were tested in vitro and several could be verified as convertible substrates. Based on information on halogenated natural products, a new dataset was created to specifically search for natural products as substrates/products, and virtual screening in this database yielded further hits.
Publikation

Sheludko, Y. V.; Volk, J.; Brandt, W.; Warzecha, H.; Expanding the diversity of plant monoterpenoid indole alkaloids employing human cytochrome P450 3A4 ChemBioChem 21, 1976-1980, (2020) DOI: 10.1002/cbic.202000020

Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R ,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.
Publikation

Schnabel, A.; Cotinguiba, F.; Athmer, B.; Yang, C.; Westermann, B.; Schaks, A.; Porzel, A.; Brandt, W.; Schumacher, F.; Vogt, T.; A piperic acid CoA ligase produces a putative precursor of piperine, the pungent principle from black pepper fruits Plant J. 102, 569-581, (2020) DOI: 10.1111/tpj.14652

Black pepper (Piper nigrum L.) is known for the high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C‐2 extension of the general C6‐C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4‐coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modeling which suggests a perfect fit of the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus‐induced gene silencing provided some preliminary indications that the production of piperoyl‐CoA is required for the biosynthesis of piperine in black pepper fruits.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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