Unexpected Methyllanthionine Stereochemistry in the Morphogenetic Lanthipeptide SapT

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) containing lanthionine (Lan) or methyllanthionine (MeLan) cross-links.
• The stereochemistry of (Me)Lan residues is critical for lanthipeptide bioactivity.
• Previously characterized lanthipeptides exhibit only 2S,3S or 2R,3R stereochemistry in their MeLan residues.

Purpose of the Study:

• To reconstitute the biosynthetic pathway of the class I lanthipeptide SapT in Escherichia coli.
• To characterize the stereochemistry of MeLan residues in heterologously produced SapT.
• To investigate the potential prevalence and origin of novel MeLan stereochemistry in other lanthipeptides.

Main Methods:

• Heterologous expression of the SapT biosynthetic pathway in E. coli.
• Synthesis of chemical standards for stereochemical analysis.
• Bioinformatic analysis of lanthipeptide biosynthetic enzymes.
• Site-directed mutagenesis to probe reaction mechanisms.

Main Results:

• Successfully reconstituted the SapT biosynthetic pathway in E. coli.
• Identified three MeLan residues in SapT with 2S,3R stereochemistry (d-allo-l-MeLan), a novel finding for lanthipeptides.
• Bioinformatic analysis and experimental verification in Streptomyces coelicolor confirmed the presence of d-allo-l-MeLan in other lanthipeptides.
• Proposed and provided evidence for a mechanism explaining the origin of this unique stereochemistry.

Conclusions:

• The discovery of d-allo-l-MeLan in SapT expands the known stereochemical diversity of lanthipeptides.
• Bioinformatic and experimental data suggest this stereochemistry is not unique to SapT and may be present in other lanthipeptides.
• The study provides insights into the enzymatic mechanisms governing MeLan formation and lanthipeptide biosynthesis.