Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis

Fredarla S Miller,Michael F Freeman,Michael F Freeman,Kathryn K Crone,Mikael H Elias,Mikael H Elias,Sudipta Shaw,Matthew R Jensen,Matthew R Jensen,Matthew R Jensen,William R Harcombe,William R Harcombe

doi:10.1038/s41467-021-25575-7

Fredarla S Miller, Michael F Freeman + Show 10 more

Open Access

https://doi.org/10.1038/s41467-021-25575-7

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Abstract

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed ‘split borosins’), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.

Highlights

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides
Translated by the ribosome, ribosomally encoded and posttranslationally modified peptides (RiPPs) are typically synthesized as short precursor peptides that are extensively posttranslationally modified at their C-termini prior to proteolytic maturation and export[1]
A new family of α-N-methylated RiPPs named the borosins was discovered as the biosynthetic origins for the nematicidal omphalotins[2,3] and the antineoplastic gymnopeptides[24], cyclic peptides produced by the basidiomycete fungi Omphalotus olearius[25,26,27] and Gymnopus fusipes[28], respectively

Summary

Introduction

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. 1234567890():,; Over the past 25 years, ribosomally synthesized and posttranslationally modified peptides (RiPPs) have proven to be a major class of natural products, whose expanding breadth of unique structures and bioactivities are beginning to rival those of non-ribosomal peptides[1] Peptide features such as amide backbone α-N-methylations[2,3] and D-configured residues[4,5,6] once thought exclusive to non-ribosomal peptides are known to be installed by RiPP biosynthetic pathways. The second distinguishing borosin family feature is that these S-adenosylmethionine-dependent (SAM-dependent) αN-methyltransferases are encoded in frame as part of unusually long leader peptides, marking the borosins as the first pathways to encode iterative, autocatalytic RiPP precursors (Fig. 1b) Soon after this discovery, the omphalotins precursor Oph(M)A30 and a close homolog[31] were structurally interrogated and found to be the first RiPP precursors to form homodimers, where each α-Nmethyltransferase acts on the C-terminal core peptide of the other precursor subunit

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