Sec Signal Peptide Doubles Up as a Leader Sequence in Bufferin Biosynthesis

The biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) typically relies on the interaction of a leader sequence at the N-terminus of the precursor peptide and a RiPP precursor recognition element (RRE). Bufferins are a newly discovered family of bacterial RiPP metallophores featuring 5-thiooxazole motifs, which are installed by a multinuclear nonheme iron-dependent oxidative enzyme (MNIO) together with its partner protein. As an original feature, bufferin precursors possess Sec-dependent signal peptides. Using a model system from Caulobacter vibrioides, this work investigated key features of bufferin biosynthesis. Biophysical characterization demonstrated the binding of the signal peptide to modification enzymes, and structural modeling showed that the interaction is via the RRE domain. This implies that the signal peptide doubles up as a leader sequence. Mutagenesis in the native C. vibrioides host indicated that the bufferin signal peptide mediates optimal coupling of post-translational modification and export to ensure bufferin function in vivo and that its composition is key in this process. Using heterologous expression in Escherichia coli, where export is bypassed, the substrate scope of the MNIO/partner pair was probed, allowing us to define features of non-native substrates. This study provides a paradigm for signal-peptide-dependent RiPP biogenesis and paves the way for bioengineering of thiooxazole-containing peptides.