@misc{YanFriemelAloisietal.2016, author = {Yan, Robert and Friemel, Martin and Aloisi, Claudia and Huynen, Martijn and Taylor, Ian A. and Leimk{\"u}hler, Silke and Pastore, Annalisa}, title = {The eukaryotic-specific Isd11 is a complex- orphan protein with ability to bind the prokaryotic IscS}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {551}, issn = {1866-8372}, doi = {10.25932/publishup-41190}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-411906}, pages = {14}, year = {2016}, abstract = {The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction.}, language = {en} } @misc{SasMuellerKappeletal.2016, author = {Sas, Claudia and M{\"u}ller, Frank and Kappel, Christian and Kent, Tyler V. and Wright, Stephen I. and Hilker, Monika and Lenhard, Michael}, title = {Repeated inactivation of the first committed enzyme underlies the loss of benzaldehyde emission after the selfing transition in Capsella}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {904}, issn = {1866-8372}, doi = {10.25932/publishup-43801}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-438018}, pages = {3313 -- 3319}, year = {2016}, abstract = {The enormous species richness of flowering plants is at least partly due to floral diversification driven by interactions between plants and their animal pollinators [1, 2]. Specific pollinator attraction relies on visual and olfactory floral cues [3-5]; floral scent can not only attract pollinators but also attract or repel herbivorous insects [6-8]. However, despite its central role for plant-animal interactions, the genetic control of floral scent production and its evolutionary modification remain incompletely understood [9-13]. Benzenoids are an important class of floral scent compounds that are generated from phenylalanine via several enzymatic pathways [14-17]. Here we address the genetic basis of the loss of floral scent associated with the transition from outbreeding to selfing in the genus Capsella. While the outbreeding C. grandiflora emits benzaldehyde as a major constituent of its floral scent, this has been lost in the selfing C. rubella. We identify the Capsella CNL1 gene encoding cinnamate: CoA ligase as responsible for this variation. Population genetic analysis indicates that CNL1 has been inactivated twice independently in C. rubella via different novel mutations to its coding sequence. Together with a recent study in Petunia [18], this identifies cinnamate: CoA ligase as an evolutionary hotspot for mutations causing the loss of benzenoid scent compounds in association with a shift in the reproductive strategy of Capsella from pollination by insects to self-fertilization.}, language = {en} } @article{ReynaGonzalezSchmidPetrasetal.2016, author = {Reyna-Gonz{\´a}lez, Emmanuel and Schmid, Bianca and Petras, Daniel and S{\"u}ssmuth, Roderich D. and Dittmann, Elke}, title = {Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries}, series = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, volume = {55}, journal = {Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1433-7851}, doi = {10.1002/anie.201604345}, pages = {9398 -- 9401}, year = {2016}, abstract = {Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases.}, language = {en} } @phdthesis{ReynaGonzalez2017, author = {Reyna Gonz{\´a}lez, Emmanuel}, title = {Engineering of the microviridin post-translational modification enzymes for the production of synthetic protease inhibitors}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-406979}, school = {Universit{\"a}t Potsdam}, pages = {XI, 91, CI}, year = {2017}, abstract = {Natural products and their derivatives have always been a source of drug leads. In particular, bacterial compounds have played an important role in drug development, for example in the field of antibiotics. A decrease in the discovery of novel leads from natural sources and the hope of finding new leads through the generation of large libraries of drug-like compounds by combinatorial chemistry aimed at specific molecular targets drove the pharmaceutical companies away from research on natural products. However, recent technological advances in genetics, bioinformatics and analytical chemistry have revived the interest in natural products. The ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of natural products generated by the action of post-translationally modifying enzymes on precursor peptides translated from mRNA by ribosomes. The great substrate promiscuity exhibited by many of the enzymes from RiPP biosynthetic pathways have led to the generation of hundreds of novel synthetic and semisynthetic variants, including variants carrying non-canonical amino acids (ncAAs). The microviridins are a family of RiPPs characterized by their atypical tricyclic structure composed of lactone and lactam rings, and their activity as serine protease inhibitors. The generalities of their biosynthetic pathway have already been described, however, the lack of information on details such as the protease responsible for cleaving off the leader peptide from the cyclic core peptide has impeded the fast and cheap production of novel microviridin variants. In the present work, knowledge on leader peptide activation of enzymes from other RiPP families has been extrapolated to the microviridin family, making it possible to bypass the need of a leader peptide. This feature allowed for the exploitation of the microviridin biosynthetic machinery for the production of novel variants through the establishment of an efficient one-pot in vitro platform. The relevance of this chemoenzymatic approach has been exemplified by the synthesis of novel potent serine protease inhibitors from both rationally-designed peptide libraries and bioinformatically predicted microviridins. Additionally, new structure-activity relationships (SARs) could be inferred by screening microviridin intermediates. The significance of this technique was further demonstrated by the simple incorporation of ncAAs into the microviridin scaffold.}, language = {en} } @article{MeyerMainzKehretal.2017, author = {Meyer, Sabine and Mainz, Andi and Kehr, Jan-Christoph and Suessmuth, Roderich and Dittmann, Elke}, title = {Prerequisites of Isopeptide Bond Formation in Microcystin Biosynthesis}, series = {ChemBioChem : a European journal of chemical biology}, volume = {18}, journal = {ChemBioChem : a European journal of chemical biology}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1439-4227}, doi = {10.1002/cbic.201700389}, pages = {2376 -- 2379}, year = {2017}, abstract = {The biosynthesis of the potent cyanobacterial hepatotoxin microcystin involves isopeptide bond formation through the carboxylic acid side chains of d-glutamate and -methyl d-aspartate. Analysis of the in vitro activation profiles of the two corresponding adenylation domains, McyE-A and McyB-A(2), either in a didomain or a tridomain context with the cognate thiolation domain and the upstream condensation domain revealed that substrate activation of both domains strictly depended on the presence of the condensation domains. We further identified two key amino acids in the binding pockets of both adenylation domains that could serve as a bioinformatic signature of isopeptide bond-forming modules incorporating d-glutamate or d-aspartate. Our findings further contribute to the understanding of the multifaceted role of condensation domains in nonribosomal peptide synthetase assembly lines.}, language = {en} }