Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• D-amino acid residues are crucial for the bioactivity of many peptides, including antibiotics and toxins.
• Ribosomally synthesized and post-translationally modified peptides (RiPPs) often contain D-amino acids.
• Radical S-adenosyl-L-methionine (SAM) enzymes are the only known biocatalysts for direct epimerization in RiPPs, but their mechanism is poorly understood.

Purpose of the Study:

• To elucidate the mechanism and structural basis of D-amino acid installation in RiPPs by radical SAM enzymes.
• To determine the atomic-resolution structure of a RiPP-modifying radical SAM enzyme with its substrate.

Main Methods:

• X-ray crystallography for atomic-resolution structure determination.
• Size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS) for structural analysis.
• Electron paramagnetic resonance (EPR) spectroscopy for mechanistic insights.
• Biochemical analyses to validate enzyme function.

Main Results:

• An atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme bound to its substrate was determined.
• Structural and biochemical data revealed the mechanism of epimerization in RiPPs.
• An unprecedented enzyme mechanism for peptide epimerization was proposed.

Conclusions:

• This study provides critical insights into how radical SAM enzymes interact with RiPPs.
• The findings advance our understanding of post-translational modifications in natural product biosynthesis.
• The elucidated mechanism offers new perspectives on radical SAM enzyme catalysis.