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Publikation

Struwe, H.; Droste, J.; Dhar, D.; Davari, M. D.; Kirschning, A.; Chemoenzymatic synthesis of a new germacrene derivative named germacrene F ChemBioChem 25, e202300599, (2024) DOI: 10.1002/cbic.202300599

The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6‐C7 to C7‐C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature‐dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror‐symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.
Publikation

Pick, L. M.; Wenzlaff, J.; Yousefi, M.; Davari, M.; Ansorge-Schumacher, M.; Lipase‐mediated conversion of protecting group silyl ethers: An unspecific side reaction ChemBioChem 24, e202300384, (2023) DOI: 10.1002/cbic.202300384

Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures alcohols through protection or deprotection with silyl groups.
Publikation

Pourhassan N., Z.; Cui, H.; Khosa, S.; Davari, M. D.; Jaeger, K.; Smits, S. H. J.; Schwaneberg, U.; Schmitt, L.; Optimized hemolysin Type 1 secretion system in Escherichia coli by directed evolution of the Hly enhancer fragment and including a terminator region ChemBioChem 23, e202100702, (2022) DOI: 10.1002/cbic.202100702

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at  five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.
Publikation

Weigel, B.; Ludwig, J.; Weber, R. A.; Ludwig, S.; Lennicke, C.; Schrank, P.; Davari, M. D.; Nagia, M.; Wessjohann, L. A.; Heterocyclic and alkyne terpenoids by terpene synthase‐mediated biotransformation of non‐natural prenyl diphosphates: Access to new fragrances and probes ChemBioChem 23, e202200211, (2022) DOI: 10.1002/cbic.202200211

Terpene synthase-mediated biotransformation of eleven synthetic sulfur- or oxygen-containing non-natural prenyl diphosphates resulted in the formation of five novel terpenoids and analogues. Uniquely, they trap intermediate steps and form heterocycles or compounds with alkyne side chains. Computational modelling differentiates convertible from inconvertible substrates and thereby provides an understanding of the detailed molecular mechanism of terpene cyclases. Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases′ ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates.
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

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

Bräuer, L.; Brandt, W.; Schulze, D.; Zakharova, S.; Wessjohann, L.; A Structural Model of the Membrane-Bound Aromatic Prenyltransferase UbiA from E. coli ChemBioChem 9, 982-992, (2008) DOI: 10.1002/cbic.200700575

We report the first reasonable model for the active site of the membrane‐bound aromatic prenyltransferase UbiA, derived from structural—not sequence—similarity to a terpene synthase, with the aid of threading, site‐directed mutagenesis, and substrate selectivities. The high similarity of the active fold of UbiA‐transferase to that of 5‐epi‐aristolochene synthase (Nictotiana tabacum ), despite a low homology, allows a hypothesis on a convergent evolution of these enzymes to be formed.
Publikation

Gromer, S.; Wessjohann, L. A.; Eubel, J.; Brandt, W.; Mutational Studies Confirm the Catalytic Triad in the Human Selenoenzyme Thioredoxin Reductase Predicted by Molecular Modeling ChemBioChem 7, 1649-1652, (2006) DOI: 10.1002/cbic.200600080

Site‐directed mutagenesis of Glu477 of the human thioredoxin reductase (see figure) to glutamine, alanine, or lysine led to a significant drop in enzymatic activity. This study reinforces previous theoretical calculations which suggested that a swapping catalytic triad exists in the active site of this enzyme.
Publikation

Brandt, W.; Dessoy, M. A.; Fulhorst, M.; Gao, W.; Zenk, M. H.; Wessjohann, L. A.; A Proposed Mechanism for the Reductive Ring Opening of the Cyclodiphosphate MEcPP, a Crucial Transformation in the New DXP/MEP Pathway to Isoprenoids Based on Modeling Studies and Feeding Experiments ChemBioChem 5, 311-323, (2004) DOI: 10.1002/cbic.200300743

Experimental and theoretical investigations concerning the second‐to‐last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4‐oxo‐DMAPP ( 12 ) and 4‐hydroxy‐DMAPP ( 11 ) were synthesized in deuterium‐ or tritium‐labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP ( 7 ), 11 , and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three‐dimensional model of the fragment L271–A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe 4 S 4 cluster, and MEcPP ( 7 ) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP ( 7 ), hydroxyl abstraction, and formation of HMBPP ( 8 ). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe 4 S 4 cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP ( 8 ) to IPP ( 9 ) and DMAPP ( 10 ) in the last step.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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