A versatile enzymatic pathway for modification of peptide C-termini

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzymology

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a diverse class of natural products.
• Daptides, a specific type of RiPP, are characterized by a C-terminal amine modification.
• The biosynthetic pathways and engineering potential of daptides are not fully understood.

Purpose of the Study:

• To establish the enzymatic requirements for daptide biosynthesis.
• To investigate the diversity and engineering potential of daptide pathways.
• To explore the substrate tolerance and modification capabilities of daptide biosynthetic enzymes.

Main Methods:

• Reconstitution of daptide biosynthesis in vitro using gene clusters from Thermobifida fusca and Streptomyces azureus.
• In vitro and in vivo characterization of YcaO enzymes in converting amine intermediates to imidazolines.
• Demonstration of enzymatic activity on modified and non-native peptide substrates.

Main Results:

• Identified sequential enzymatic steps: oxidative decarboxylation, transamination, and N,N-dimethylation.
• Confirmed YcaO enzymes catalyze the formation of C-terminal imidazolines from secondary amine intermediates.
• Showcased broad substrate tolerance, enabling activity on leader peptide-free and non-native core peptides.
• Successfully engineered the daptide pathway to install new C-termini, including aminoacetone, onto various peptide and protein substrates.

Conclusions:

• The study defines the core enzymatic machinery and requirements for daptide biosynthesis.
• Daptide biosynthetic enzymes exhibit significant substrate flexibility, opening avenues for pathway engineering.
• This research provides a foundation for developing novel peptide modifications and bioconjugation strategies using engineered daptide pathways.