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

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) biosynthesis involves leader sequences and RiPP precursor recognition elements (RREs).
• Bufferins are novel bacterial RiPP metallophores characterized by 5-thiooxazole motifs installed by a multinuclear nonheme iron-dependent oxidative enzyme (MNIO) and partner protein.
• Bufferin precursors uniquely feature Sec-dependent signal peptides, distinguishing them from typical RiPPs.

Purpose of the Study:

• To investigate the key features of bufferin biosynthesis using a model system from *Caulobacter vibrioides*.
• To determine the role of the signal peptide in bufferin post-translational modification and export.
• To define the substrate scope of the MNIO/partner enzyme pair for bioengineering applications.

Main Methods:

• Biophysical characterization to analyze signal peptide-modification enzyme interactions.
• Structural modeling to elucidate the binding interface between the signal peptide and the RRE domain.
• Site-directed mutagenesis in *C. vibrioides* to assess the in vivo function of the signal peptide.
• Heterologous expression in *Escherichia coli* to probe enzyme substrate specificity.

Main Results:

• Biophysical data confirmed signal peptide binding to modification enzymes via the RRE domain, suggesting the signal peptide functions as a leader sequence.
• Mutagenesis studies revealed that the bufferin signal peptide is crucial for coordinating post-translational modification and export, ensuring in vivo bufferin function.
• Heterologous expression experiments successfully characterized the substrate scope of the MNIO/partner enzyme complex, identifying features of non-native substrates.

Conclusions:

• The signal peptide in bufferin biosynthesis acts as a leader sequence, essential for efficient post-translational modification and export.
• This study establishes a novel paradigm for signal-peptide-dependent RiPP biogenesis.
• The findings provide a foundation for the bioengineering of peptides containing thiooxazole motifs.