Untersuchungen zur Biosynthese von ribosomal synthetisierten Lassopeptiden und makrozyklischen Peptidantibiotika

• Antragsteller: Professor Dr. Mohamed A. Marahiel
• Fachliche Zuordnung: Biochemie
• Förderung: Förderung von 2010 bis 2015
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 178860017

• Erstellungsjahr: 2016

Zusammenfassung der Projektergebnisse

This project was dedicated towards the understanding of the biochemical and structural aspects of novel class of ribosomally synthesized peptides antibiotics with a focus on bacterial lasso peptides and other macrocyclic peptides such as sactipeptides and cyclic-dipeptides. Applying a novel genome mining approach for lasso peptide discovery we were able to identify over 100 different gene clusters from a total of 87 different proteobacterial strains. Over 20 new lasso peptides were expressed in heterologous systems, purified and their specific MS-fragmentation patterns and some NMR-structures were elucidated. We have also in collaboration biochemically explored the roles of the ATP-dependent cysteine protease (protein B, associated with leader peptide release and folding) and the adenylate-forming enzyme (protein C, associated with acidic site chain activation and isopeptide bond formation) in the microcin J25 system, which represents a model system for lasso peptide maturation. In addition we discovered in this study a new 7-residues ring lasso peptides besides the well-known 8- and 9-membered ring structures and used the lasso peptide scaffold for peptide grafting for the first time. For the ribosomally synthesized sactipeptides (subtilosin A, Thurincin H and the sporulation killing factor) we explored their unique enzymatic mechanism for the formation of the thioether bond formation (post-translational modification) by SAM-radical enzymes. For the class of the ribosomally synthesized cyclic dipeptides that show the privileged 2, 5-diketopiperazine (DKP) scaffold, we explored in this study some of their modifying enzymes that are responsible for installing functional groups crucial for the biological activity of the resulting modified DKPs. The results obtained lead to important discoveries in the fascinating fields of lasso peptide structural elucidation, biosynthesis and grafting as well as to important insights into the mechanisms of post-translational modification of other ribosomally synthesized macrocyclic peptides.

Projektbezogene Publikationen (Auswahl)

• (2010) The glucagon receptor antagonist I-32169 constitutes a new class of lasso peptides. FEBS Lett. 584: 785-789
  Knappe, T.A., Linne, U., Xie, X. and Marahiel, M.
  
• (2011) Introducing lasso peptides as molecular scaffolds for drug design: engineering of an integrin antagonist. Angew. Chem. Int. Ed. Engl. 50, 8714-8717
  Knappe, T.A., Manzenrieder, F., Mas-Moruno, C., Linne, U., Sasse, F., Kessler, H., Xie, X. and Marahiel, M.A.
  
• (2011) The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase. J. Mol. Biol. 412, 842-848
  Kuznedelov, K., Semenova, E., Knappe, T.A., Mukhamedyarov, D., Srivstava, A., Chatterjee, S., Ebright, R.H., Marahiel, M.A. and Severinov, K.
  
• (2011) Topoisomer differentiation of molecular knots by FTICR MS: Lessons from class II Lasso peptides. J. Am. Soc. Mass. Spectrom. 22, 467-479
  Zirah, S., Afonso, C., Linne, U., Knappe, T., Marahiel, M. Rebuffat, S. and Tabet, J.-C.
  
• (2012) Dissecting the maturation steps of the lasso peptide microcin J25 in vitro. Chembiochem 13 1046-1052
  Yan, K.P., Li, Y., Zirah, S., Goulard, C., Knappe, T.A., Marahiel, M.A. and Rebuffat, S.
  
• (2012) NMR as an effective tool for the structure determination of lasso peptides. Chembiochem 13, 621-625
  Xie, X. and Marahiel, M.A.
  
• (2012) Ribosome-independent biosynthesis of biologically active peptides: Application of synthetic biology to generate structural diversity. FEBS Lett. 586, 2065-2075
  Giessen, T.W. and Marahiel, M.A.
  
• (2012) The radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A. Nat. Chem. Biol. 8 350-357
  Flühe, L., Knappe, T.A., Gattner, M.J., Schäfer, A., Burghaus, O., Linne, U. and Marahiel, M.A.
  
• (2013) A tRNA-dependent two-enzyme pathway for the generation of singly and doubly methylated ditryptophan 2,5-diketopiperazines. Biochemistry 52, 4274-4283
  Giessen, T.W., von Tesmar, A.M. and Marahiel, M.A.
  
• (2013) Caulosegnins I-III: A highly diverse group of lasso peptides derived from a single biosynthetic gene cluster. J. Am. Chem. Soc. 135 210-222
  Hegemann, J.D., Zimmermann, M., Xie, X. and Marahiel, M.A.
  
• (2013) Insights into the generation of structural diversity in a tRNA-dependent pathway for highly modified bioactive cyclic dipeptides. Chemistry & Biology 20, 828-838
  Giessen, T.W., von Tesmar, A.M. and Marahiel, M.A.
  
• (2013) Lasso peptides from proteobacteria: Genome mining employing heterologous expression and mass spectrometry. Biopolymers 100, 527-542
  Hegemann, J.D., Zimmermann, M., Zhu, S., Klug, D. and Marahiel, M.A.
  
• (2013) The astexin-1 lasso peptides: Biosynthesis, stability, and structural studies. Chemistry & Biology 20 558-569
  Zimmermann, M., Hegemann, J.D., Xie, X. and Marahiel, M.A.
  
• (2013) Two [4Fe-4S] clusters containing radical SAM enzyme SkfB catalyze thioether bond formation during the maturation of the sporulation killing factor. J. Am. Chem. Soc. 135 959-962
  Flühe, L., Burghaus, O., Wieckowski, B.M., Giessen T.W., Linne, U. and Marahiel, M.A.
  
• (2014) Characterization of caulonodin lasso peptides revealed unprecedented N-terminal residues and a precursor motif essential for peptide maturation. Chemical Sience 5, 4032-4043
  Zimmermann, M., Hegemann, J.D., Xie, X. and Marahiel, M.A.
  
• (2014) Rational improvement of the affinity and selectivity of integrin binding of grafted lasso peptides. J. Med. Chem. 57, 5829-5834
  Hegemann, J.D., De Simone, M., Zimmermann, M., Knappe, T., Xie, X., Di Leva, F.S., Marinelli, L., Novellino, E., Zahler, S., Kessler, H. and Marahiel, M.A.
  
• (2014) Xanthomonins I-III: A new class of lasso peptides with a seven-residue macrolactam ring. Angewandte Chemie 126, 2230-2234
  Hegemann, J.D., Zimmermann, M., Zhu, S., Steuber, H., Harms, K., Xie, X. and Marahiel, M.A.
  
• (2015) Ion mobility-mass spectrometry of lasso peptides: signature of a rotaxane topology. Anal. Chem. 87, 1166-1172
  Fouque, J.D., Afonso, C., Zirah, S., Hegemann, J.D., Zimmermann, M., Marahiel, M.A., Rebuffat, S. and Lavanant, H.
  
• (2015) The PqqD homologous domain of the radical SAM enzyme ThnB is required for thioether bond formation during thurincin H maturation. FEBS Lett. 1802-1806
  Wieckowski, B.M., Hegemann, J.D., Mielcarek, A., Boss, L., Burghaus, O. and Marahiel, M.A.