Substrate-Controlled Catalysis in the Ether Cross-Link-Forming Radical SAM Enzymes

Area of Science:

• Biochemistry
• Natural Product Biosynthesis
• Enzymology

Background:

• Darobactin is a complex heptapeptide antibiotic.
• It features both ether and C-C cross-links.
• The enzyme responsible, DarE, is a radical S-adenosylmethionine (rSAM) enzyme, but how it forms both cross-links is unclear.

Purpose of the Study:

• To investigate the biosynthetic pathways of darobactin-like RiPPs (ribosomally synthesized and post-translationally modified peptides).
• To understand the enzymatic mechanisms underlying cross-link formation by rSAM enzymes.
• To discover novel daropeptides and elucidate the factors controlling their structural diversity.

Main Methods:

• Biosynthetic gene cluster analysis.
• Phylogenetic analysis of maturase enzymes.
• Site-directed mutagenesis of key residues in daropeptide maturases.
• Biochemical characterization of enzyme activity.

Main Results:

• Identification and characterization of two novel daropeptides containing only ether cross-links.
• Demonstration that daropeptide maturases possess intrinsic multifunctionality.
• Evidence that substrate-specific motifs dictate the formation of ether cross-links, C-C cross-links, and Ser oxidation.
• rSAM enzymes can catalyze distinct chemical transformations based on substrate recognition.

Conclusions:

• Daropeptide maturases are versatile enzymes capable of catalyzing multiple types of post-translational modifications.
• Substrate sequence directly influences the chemical outcome of rSAM enzyme activity.
• This work provides a framework for discovering new RiPPs and understanding the regulation of rSAM enzyme multifunctionality.