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Structural and functional analysis of non-canonical tandem-thioesterases
Nonribosomal peptide synthetases (NRPS) are known to produce a diverse range of bioactive natural products, like antibiotics, immunosuppressants or cytostatics. These pharmacologically important properties have attracted increased scientific interest, on the one hand to advance the discovery of new...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2021
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| Online Zugang: | PDF-Volltext |
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| Zusammenfassung: | Nonribosomal peptide synthetases (NRPS) are known to produce a diverse range of bioactive natural products, like antibiotics, immunosuppressants or cytostatics. These pharmacologically important properties have attracted increased scientific interest, on the one hand to advance the discovery of new natural products from nature. On the other hand, the mechanistic elucidation of these complex synthetic pathways enables the catalytic properties of individual domains to be used for targeted drug design. Regarding mechanistic elucidation, research is particularly dependent on the structural elucidation of individual domains and modules in order to reveal functionally essential interactions, to investigate substrate specificities and facilitate the targeted modification of certain functional units. A particular interest here is the functional analysis of termination units, such as thioesterase (Te), which is indispensable for product release from the NRPS. In recent years, the interest has grown in particular in the study of the unusual tandem Te, which has been described predominantly in NRPS systems whose biosynthesis seem to represent predominantly macrocyclic natural products.
In this work, the structural and functional analysis of unusual tandem Te based on the model organism Xenorhabdus nematophila ATCC 19061 is described. Previous work by BODE et al. has already shown that the biosynthetic gene cluster of xenoamicin, a member of the cyclic depsipeptide group, harbors this didomain. One of the focal questions of this work deals with the structure elucidation of this tandem-XncTe, which is described in more detail here at a resolution of 3.4 Å. In addition, a C-terminal subdomain is revealed at a resolution of 1.9 Å, reveals properties of the isolated subdomains. The structural information is also linked to dynamic properties in solution, which are also spectrometrically classified via hydrogen/deuterium exchange. Here, the focus relies on the subdomain arrangement in particular, as well as Te-known lid features. In a further bioinformatic approach, the natural occurrence of tandem-Te is also highlighted and illuminated in greater depth using collected sequence information.
A further part of this paper, the protein biochemical characterization of the orthologous 2-F4 NRPS tandem Te of unknown origin is also described. The ortholog, which shares similarity to the syringomycin synthetase from P. syringae, is successfully recombinantly overproduced and purified for the first time. Based on this, the solubilized C-terminal partial structure is presented, which shows a strongly conserved structure to tandem Xnc-Te. The first dynamic investigations could also be carried out here. |
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| Umfang: | 237 Seiten |
| DOI: | 10.17192/z2021.0493 |