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) are a diverse class of natural products.
• Daptides, a subclass of RiPPs, are characterized by a C-terminal amine instead of a carboxylic acid.
• The biosynthetic pathways and engineering potential of daptides are not fully understood.

Purpose of the Study:

• To establish the diversity, enzymatic requirements, and engineering potential of daptide biosynthesis.
• To reconstitute and characterize the key enzymatic steps in daptide formation.
• To explore the substrate tolerance and engineering capabilities of the daptide biosynthetic machinery.

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 C-terminal imidazolines.
• Demonstration of enzymatic activity on modified and non-native peptide substrates.

Main Results:

• The study identified sequential enzymatic requirements for daptide biosynthesis: oxidative decarboxylation, transamination, and N,N-dimethylation.
• A family of YcaO enzymes was shown to catalyze the final conversion of a secondary amine to a C-terminal imidazoline.
• The daptide pathway enzymes exhibited broad substrate tolerance, accepting shortened, leader peptide-free, and non-native core peptides.
• Engineered daptide pathways successfully installed novel C-termini, including an aminoacetone moiety, onto various peptide and protein substrates.

Conclusions:

• The enzymatic machinery for daptide biosynthesis has been elucidated, revealing key steps and enzyme families involved.
• The broad substrate tolerance of daptide pathway enzymes offers significant potential for synthetic biology applications.
• This work enables the engineering of novel peptide and protein C-termini for applications such as bioconjugation.