Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs.
|Original language||English (US)|
|Title of host publication||Synthetic and Enzymatic Modifications of the Peptide Backbone|
|Editors||E. James Petersson|
|Publisher||Academic Press Inc.|
|Number of pages||30|
|State||Published - Jan 2021|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
The authors thank Jörn Piel and his team at ETH Zürich for helpful discussions and providing access to their HPLC-MS/MS systems as well as technical expertise. This work was financially supported by ETH Zürich and the Swiss National Science Foundation (Grant no. 31003A_173097 to MK).
© 2021 Elsevier Inc.
- Protein Processing, Post-Translational
PubMed: MeSH publication types
- Research Support, Non-U.S. Gov't
- Journal Article