Structural, Biochemical, and Bioinformatic Basis for Identifying Radical SAM Cyclopropyl Synthases

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Radical S-adenosyl-l-methionine (RS) enzymes, particularly the RS-SPASM subfamily, are vital for modifying peptides.
• Cyclopropane scaffolds are important in pharmaceutical development, necessitating a deeper understanding of their synthesis.

Purpose of the Study:

• To define the family of cyclopropyl (CP) synthases.
• To characterize a novel CP synthase, TigE, and its associated RiPP pathway.
• To elucidate the structural and mechanistic basis of CP synthase activity.

Main Methods:

• Bioinformatic expansion of CP synthases using RadicalSAM.org.
• Identification and characterization of the TigB precursor peptide and TigE enzyme.
• Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses.
• X-ray crystallography of TigE.

Main Results:

• A novel RiPP pathway involving the TigB peptide with a repeating TIGSVS motif was identified.
• TigE was shown to catalyze the formation of methyl-cyclopropylglycine (methyl-CPG) from isoleucine within TigB.
• The crystal structure of TigE revealed a unique glycine-tyrosine-tryptophan motif enabling tyrosyl ligation to the [4Fe-4S] cluster, essential for activity.

Conclusions:

• The study defines the CP synthase family and characterizes a novel member, TigE.
• A unique mechanism involving tyrosyl ligation to an auxiliary cluster is crucial for CP synthase function.
• These findings provide insights into the biosynthesis of cyclopropane-containing peptides.