N-methylated peptide macrocycle omphalotin A (nematicidal cyclododecapeptide). A methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Fungi: NE > Dikarya: NE > Basidiomycota: NE > Agaricomycotina: NE > Agaricomycetes: NE > Agaricomycetidae: NE > Agaricales: NE > Marasmiineae: NE > Omphalotaceae: NE > Omphalotus: NE > Omphalotus olearius: NE
Molecular evidence
Database
No mutation 4 structures(e.g. : 7ZAZ, 7ZB0, 7ZB1... more)(less) 7ZAZ: Omphalotus olearius macrocyclase OphP with ZPP, 7ZB0: Omphalotus olearius macrocyclase OphP with 15mer, 7ZB1: Omphalotus olearius macrocyclase OphP S580A with 18mer, 7ZB2: Omphalotus olearius macrocyclase OphP S580A apo No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MSFPGWGPYPPVERDETSAITYSSKLHGSVTVRDPYSQLEVPFEDSEETK AFVHSQRKFARTYLDENPDREAWLETLKKSWNYRRFSALKPESDGHYYFE YNDGLQSQLSLYRVRMGEEDTVLTESGPGGELFFNPNLLSLDGNAALTGF VMSPCGNYWAYGVSEHGSDWMSIYVRKTSSPHLPSQERGKDPGRMNDKIR HVRFFIVSWTSDSKGFFYSRYPPEDDEGKGNAPAMNCMVYYHRIGEDQES DVLVHEDPEHPFWISSVQLTPSGRYILFAASRDASHTQLVKIADLHENDI GTNMKWKNLHDPWEARFTIVGDEGSKIYFMTNLKAKNYKVATFDANHPDE GLTTLIAEDPNAFLVSASIHAQDKLLLVYLRNASHEIHIRDLTTGKPLGR IFEDLLGQFMVSGRRQDNDIFVLFSSFLSPGTVYRYTFGEEKGYRSLFRA ISIPGLNLDDFMTESVFYPSKDGTSVHMFITRPKDVLLDGTSPVLQYGYG GFSLAMLPTFSLSTLLFCKIYRAIYAIPNIRGGSEYGESWHREGMLDKKQ NVFDDFNAATEWLIANKYASKDRIAIRGGSNGGVLTTACANQAPGLYRCV ITIEGIIDMLRFPKFTFGASWRSEYGDPEDPEDFDFIFKYSPYHNIPPPG DTVMPAMLFFTAAYDDRVSPLHTFKHVAALQHNFPKGPNPCLMRIDLNSG HFAGKSTQEMLEETADEYSFIGKSMGLTMQTQGSVDSSRWSCVTV
The enzyme OphP is essential for the biosynthesis of the macrocyclic peptide omphalotin A, a dodecamer with 9 backbone N-methylations produced by the wood-degrading fungus Omphalotus olearius. Heterologous expression of OphP and the peptide-precursor protein OphMA in yeast, yields omphalotin A. Thus, Oph P was hypothesized to have a dual function; catalyzing both endoproteolytic release of a peptide intermediate from OphMA, and macrocyclization of the multiply alpha-N-methylated core peptide with concomitant release of a C-terminal follower peptide. In our in vitro activity assays, OphP showed robust endoproteolytic and macrocyclase activity on alpha-N-methylated peptides but was unable to cleave OphMA. The enzyme had a strong preference for hydrophobic, highly alpha-N-methylated peptides and an alpha-N-methylated glycine residue at the P1 site. OphP adopts a canonical prolyl oligopeptidase (POP) fold with a predominantly hydrophobic substrate binding cleft, and a small and hydrophobic P1 binding pocket. We demonstrate that OphP is a POP-type macrocyclase with a specificity and a substrate route to the active site different from other members of the family. These results could be exploited for the biotechnological production of macrocyclic peptides with multiple backbone N-methylations, which are interesting due to their favorable pharmacological properties.
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 alpha-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.
Omphalotus olearius (Jack o'lantern) macrocyclase OphP prolyl oligopeptidase
