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Publikation

Gumz, F.; Krausze, J.; Eisenschmidt, D.; Backenköhler, A.; Barleben, L.; Brandt, W.; Wittstock, U.; The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown Plant Mol. Biol. 89, 67-81, (2015) DOI: 10.1007/s11103-015-0351-9

Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide “top” and a narrower “bottom” opening with distinct strand-connecting loops protruding far beyond the lower propeller surface. Molecular modeling and mutational analysis identified residues for glucosinolate aglucone and Fe2+ cofactor binding within these loops. As the first experimentally determined structure of a plant kelch protein, the crystal structure of TaTFP not only enables more detailed mechanistic studies on glucosinolate breakdown product formation, but also provides a new basis for research on the diverse roles and mechanisms of other kelch proteins in plants.
Publikation

Farag, M. A.; Al-Mahdy, D. A.; Salah El Dine, R.; Fahmy, S.; Yassin, A.; Porzel, A.; Brandt, W.; Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen Chem. Biodivers. 12, 955-962, (2015) DOI: 10.1002/cbdv.201400194

Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc‐zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity‐guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5‐dihydroxy‐4‐methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5–9 mg/ml), with 3,5‐dihydroxy‐4‐methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure–activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids.
Publikation

Dippe, M.; Brandt, W.; Rost, H.; Porzel, A.; Schmidt, J.; Wessjohann, L. A.; Rationally engineered variants of S-adenosylmethionine (SAM) synthase: reduced product inhibition and synthesis of artificial cofactor homologues Chem. Commun. 51, 3637-3640, (2015) DOI: 10.1039/c4cc08478k

S-Adenosylmethionine (SAM) synthase was engineered for biocatalytic production of SAM and long-chain analogues by rational re-design. Substitution of two conserved isoleucine residues extended the substrate spectrum of the enzyme to artificial S-alkylhomocysteines. The variants proved to be beneficial in preparative synthesis of SAM (and analogues) due to a much reduced product inhibition.
Publikation

Brandt, W.; Manke, K.; Vogt, T.; A catalytic triad – Lys-Asn-Asp – Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases Phytochemistry 113, 130-139, (2015) DOI: 10.1016/j.phytochem.2014.12.018

Crystal structure data of cation-dependent catechol O-methyltransferases (COMTs) from mammals and related caffeoyl coenzyme A OMTs (CCoAOMTs) from plants have suggested operative molecular mechanisms. These include bivalent cations that facilitate deprotonation of vicinal aromatic dihydroxy systems and illustrate a conserved arrangement of hydroxyl and carboxyl ligands consistent with the requirements of a metal-activated catalytic mechanism. The general concept of metal-dependent deprotonation via a complexed aspartate is only one part of a more pronounced proton relay, as shown by semiempirical and DFT quantum mechanical calculations and experimental validations. A previously undetected catalytic triad, consisting of Lys157-Asn181-Asp228 residues is required for complete methyl transfer in case of a cation-dependent phenylpropanoid and flavonoid OMT, as described in this report. This triad appears essential for efficient methyl transfer to catechol-like hydroxyl group in phenolics. The observation is consistent with a catalytic lysine in the case of mammalian COMTs, but jettisons existing assumptions on the initial abstraction of the meta-hydroxyl proton to the metal stabilizing Asp154 (PFOMT) or comparable Asp-carboxyl groups in type of cation-dependent enzymes in plants. The triad is conserved among all characterized plant CCoAOMT-like enzymes, which are required not only for methylation of soluble phenylpropanoids like coumarins or monolignol monomers, but is also present in the similar microbial and mammalian cation-dependent enzymes which methylate a comparable set of substrates.
Publikation

Bobach, C.; Tennstedt, S.; Palberg, K.; Denkert, A.; Brandt, W.; de Meijere, A.; Seliger, B.; Wessjohann, L. A.; Screening of synthetic and natural product databases: Identification of novel androgens and antiandrogens Eur. J. Med. Chem. 90, 267-279, (2015) DOI: 10.1016/j.ejmech.2014.11.026

The androgen receptor is an important pharmaceutical target for a variety of diseases. This paper presents an in silico/in vitro screening procedure to identify new androgen receptor ligands. The two-step virtual screening procedure uses a three-dimensional pharmacophore model and a docking/scoring routine. About 39,000 filtered compounds were docked with PLANTS and scored by Chemplp. Subsequent to virtual screening, 94 compounds, including 28 steroidal and 66 nonsteroidal compounds, were tested by an androgen receptor fluorescence polarization ligand displacement assay. As a result, 30 compounds were identified that show a relative binding affinity of more than 50% in comparison to 100 nM dihydrotestosterone and were classified as androgen receptor binders. For 11 androgen receptor binders of interest IC50 and Ki values were determined. The compound with the highest affinity exhibits a Ki value of 10.8 nM. Subsequent testing of the 11 compounds in a PC-3 and LNCaP multi readout proliferation assay provides insights into the potential mode of action. Further steroid receptor ligand displacement assays and docking studies on estrogen receptors α and β, glucocorticoid receptor, and progesterone receptor gave information about the specificity of the 11 most active compounds.
Publikation

Khalil, M. N.; Brandt, W.; Beuerle, T.; Reckwell, D.; Groeneveld, J.; Hänsch, R.; Gaid, M. M.; Liu, B.; Beerhues, L.; O-Methyltransferases involved in biphenyl and dibenzofuran biosynthesis Plant J. 83, 263-276, (2015) DOI: 10.1111/tpj.12885

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O‐methylation reactions. cDNAs encoding the O‐methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab‐causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5‐dihydroxybiphenyl, supplied by the first pathway‐specific enzyme, biphenyl synthase (BIS). 3,5‐Dihydroxybiphenyl underwent a single methylation reaction in the presence of S‐adenosyl‐l‐methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5‐hydroxyferulic acid. Both substrates were only methylated at the meta‐positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor‐treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N‐ and C‐terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.
Publikation

Heinze, M.; Brandt, W.; Marillonnet, S.; Roos, W.; “Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints Plant Cell 27, 448-462, (2015) DOI: 10.1105/tpc.114.135343

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.
Publikation

Yahyaa, M.; Matsuba, Y.; Brandt, W.; Doron-Faigenboim, A.; Bar, E.; McClain, A.; Davidovich-Rikanati, R.; Lewinsohn, E.; Pichersky, E.; Ibdah, M.; Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot Plant Physiol. 169, 1683-1697, (2015) DOI: 10.1104/pp.15.00930

Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms.
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

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”.
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