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Projekt Druckansicht

Identifizierung eines generellen Biosyntheseweges von 3-Acyltetronaten und weitergehende Untersuchung des Biosyntheseweges der ionophoren Polyether Tetronasin und Tetronomycin auf Proteinebene

Antragsteller Professor Dr. Frank Hahn
Fachliche Zuordnung Biologische und Biomimetische Chemie
Förderung Förderung von 2009 bis 2010
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 112065983
 
Erstellungsjahr 2010

Zusammenfassung der Projektergebnisse

The main aim of the project, the 'Identification of a General Biosynthetic Pathway of 3-Acyltetronates' was accomplished. rkD from the RK-682 gene cluster was found to code for an FabH-like protein RkD, which catalyses the conversion of the biosynthetic precursors into the tetronate-containing natural product RK-682. In the course of the project an in vitro assay was developed, which proved that RkD is solely required to join together glyceroyl-S-ACP thioester and β-keto-S-ACP thioester and ultimately form a tetronate ring. This was the first time that an enzyme was unambigiously identified to catalyse tetronate formation. The results of this work were published in Nature Chemical Biology.5 FabH-like proteins were also found in the gene clusters of tetronasin and tetronomycin but could not yet be heterologously expressed in a soluble form. A synthetic route was developed, which gives general access to rather hardly accessible β-keto-CoA esters. These can be transfered into β-keto-S-ACP thioesters by reaction with the broad specific phosphopantheteinyl transferase Sfp. Some of these β-keto CoA esters were applied in the tetronate forming assay. It showed that RkD is substrate tolerant and it also accepts β-keto-S-ACP thioesters with much shorter chains than the native precursor. This high substrate tolerance was rather unexpected, because FabH enzymes are usually keyhole enzymes in fatty acid biosynthesis and thus restricted to accept only very defined chain lengths. As a consequence, it can be assumed that mutagenesis at key positions could further expand the substrate specificity. From the information gained during the project, it became clear that the minimum chain length that is accepted by the native enzyme is between three and seven carbons long. Further work will concentrate on the investigation of the substrate specificity of this enzyme. Interesting chain modifications would for example be unsaturations, cycles and groups like azides that enable the coupling via bioorthogonal reactions like Click-Chemistry or the Staudinger reaction. Preliminary work on the biosynthesis of tetrahydrofuran rings in tetronasin and tetronomycin was also accomplished. The enzymes TsnB and TsnC were heterologously expressed and the expression plasmids for TmnB and TmnC were prepared. Initial attempts to a synthetic route to precursors were also examined and will serve as the starting point for further optimisations.

Projektbezogene Publikationen (Auswahl)

  • Mechanistic insight into tetronate ring formation from in vitro reconstruction of the entire pathway to the cell cycle protein phosphatase inhibitor RK-682; Nature Chemical Biology
    Yuhui Sun, Frank Hahn, Yuliya Demydchuk, James Chettle, Manuela Tosin, Hiroyuki Osada and Peter F. Leadlay
    (Siehe online unter https://doi.org/10.1038/nchembio.285)
 
 

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