Methylene Peptide Backbone Substitution Significantly Affects the Glycopeptide Antibiotic Cross-Linking Cascade

Area of Science:

• Biochemistry
• Molecular Biology
• Medicinal Chemistry

Background:

• Glycopeptide antibiotics (GPAs) possess complex cross-linked structures essential for their function.
• Cytochrome P450 (Oxy) enzymes and their X-domain facilitate the installation of these cross-links.
• Understanding Oxy enzyme substrate tolerance is key to exploring GPA synthetic complexity and antibiotic activity.

Purpose of the Study:

• To investigate the impact of amide backbone modifications on Oxy enzyme-mediated cross-linking in GPAs.
• To assess the tolerance of Oxy enzymes for a specific [Ψ[CH2NH]Tpg] methylene linkage in peptide substrates.

Main Methods:

• Synthesized GPA peptide analogues containing a methylene linkage in the amide backbone.
• Assessed the ability of Oxy enzymes to perform cross-linking on these modified substrates.

Main Results:

• Oxy enzymes exhibited extreme sensitivity to the introduced methylene linkage.
• The presence of the methylene linkage significantly hindered or prevented the cross-linking cascade.

Conclusions:

• Backbone carbonyl groups are critical for proper peptide substrate binding to Oxy enzymes during GPA biosynthesis.
• Oxy enzyme sensitivity to backbone modifications highlights a key limitation in engineering novel GPA structures.