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By Francesca Mosca (IBS/Biomolecular NMR Spectroscopy Group)

This thesis investigated the structure and function of L,D-transpeptidases (LDTs) responsible for covalently attaching Braun’s lipoprotein (Lpp) to the peptidoglycan layer in Gram-negative bacteria. Because Lpp constitutes the principal covalent connection between the peptidoglycan and the outer membrane, disruption of this linkage destabilises the bacterial cell envelope. Understanding the molecular mechanisms underlying LDT-mediated Lpp attachment therefore provides a foundation for developing novel inhibitors capable of compromising outer-membrane integrity and enhancing the activity of existing antibiotics against antibiotic-resistant Gram-negative bacteria. The work focused on mainly two aspects of LDT function: the interactions of these enzymes with existing and novel antibiotics, and with their physiological substrates Lpp and peptidoglycan. Particular emphasis was placed on LdtB, the most essential LDT in E. coli. Structural and functional characterization was performed using NMR spectroscopy, biophysical methods, and biochemical techniques, complemented by in silico approaches, including molecular dynamics simulations.This integrative approach was used to decipher the molecular determinants of Lpp-peptidoglycan transpeptidation mechanism and to propose a possible stuctural model for the reaction, which remains poorly understood despite its biological relevance. In this context, E. coli served as a model system, whose study may provide insights into mechanisms that could be conserved across other Gram-negative bacteria. This work was carried out within the framework of the BREAKthrough consortium, which enabled part of the research to be conducted in collaboration with partner laboratories across Europe.