In addition to their role in protein synthesis by the ribosomes, aminoacyl-tRNAs participate in various metabolic pathways as a source of ester-activated amino acids. Among the tRNA-dependent aminoacyl transferases, enzymes of the Fem family catalyze an essential step of peptidoglycan synthesis in pathogenic bacteria and are considered as attractive targets for the development of novel antibiotics. FemX, the model enzyme of the family, transfers L-Ala from Ala-tRNA to the epsilon-amino group of L-Lys in the peptidoglycan precursor UDP-MurNAc-pentapeptide (UM5K). The crystal structures of the apo-enzyme and of a UM5K-FemX complex have been determined but co-crystallization with Ala-tRNA has not been obtained. We propose to develop the semi-synthesis of highly modified aminoacyl-tRNAs and bi-substrates to explore the catalytic mechanism of FemX. We will synthesize chemical probes that will specifically interact with FemX and its substrates. Azides and alkynes will be introduced into the tRNA and in UM5K, respectively. The Huisgen-Sharpless Cu(I)-catalyzed cycloaddition reaction will afford bi-substrates containing the tRNA covalently linked to the peptidoglycan precursor. In parallel, the active center of FemX will be used to catalyze the same reaction. By this approach, we will obtain molecules suitable for co-crystallization with FemX. Because the in situ generated reaction products are likely to trap a single conformational state of FemX corresponding to the catalytically active form of the enzyme, this approach is likely to be more powerful than the conventional crystallogenesis screens made with the substrates or products of the reaction. Phospho-derivatives of the tRNA will be synthesized to mimic the putative tetrahedral intermediate resulting from the intramolecular nucleophilic attack of the carbonyl of Ala-tRNA by the vicinal ribose hydroxyl. These phospho-derivatives will also be used to trap a relevant conformation of the enzyme that allows the trans-acylation reaction of the amino acid between the 2’ and 3’ positions of Ala-tRNA to occur within the active site. The enzyme-catalyzed cycloaddition reaction will be further investigated both to identify inhibitors of FemX and to decipher the mechanism of the enzyme-assisted catalyzed cycloaddition reaction, which is poorly understood. We will assess and compare the contributions of substrate binding and substrate activation (i) in the CuI- and FemX-catalyzed cycloaddition reactions using the functionalized substrates, and (ii) in the amino acid transfer reaction catalyzed by FemX with the “natural” substrates. The information gathered on the catalytic mechanism of FemX and on the structure of its active site should provide the critical information for the rationale design of drugs active on Fem transferases from pathogenic bacteria such as methicillin-resistant staphylococci. The approach will be of broad application in RNA biology.