Mechanistic studies on the substrate-tolerant lanthipeptide synthetase ProcM

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Synthesis

Background:

• Lanthipeptides are modified peptides with diverse biological activities.
• Lanthionine (Lan) and methyllanthionine (MeLan) residues are key structural features.
• The lanthipeptide synthetase ProcM exhibits high substrate tolerance, posing mechanistic questions.

Purpose of the Study:

• To elucidate the catalytic mechanism of the substrate-tolerant lanthipeptide synthetase ProcM.
• To investigate the order of dehydration and cyclization steps in lanthipeptide synthesis.
• To explore the basis of ProcM's broad substrate specificity.

Main Methods:

• A hybrid ligation strategy combining expressed protein ligation and copper-catalyzed azide-alkyne cycloaddition was employed.
• Substrate analogues with deuterium-labeled amino acids and orthogonal cysteine protection were synthesized.
• Analysis of product formation and proton exchange was performed to understand enzyme activity.

Main Results:

• Dehydration and cyclization by ProcM occur in a specific C-to-N-terminal order, dependent on substrate sequence.
• Non-enzymatic cyclization was ruled out as a factor in ProcM's high substrate tolerance.
• Evidence suggests that ProcM-mediated ring formation may be reversible, indicated by proton exchange at MeLan.

Conclusions:

• The study provides detailed mechanistic insights into the substrate-tolerant ProcM.
• Understanding ProcM's mechanism can guide future engineering of lanthipeptides.
• The developed hybrid ligation strategy is valuable for synthesizing lanthipeptide analogues.