Identification of the UDP-MurNAc-Pentapeptide:l-Alanine Ligase for Synthesis of Branched Peptidoglycan Precursors in Enterococcus faecalis

Many species of gram-positive bacteria produce branched peptidoglycan precursors resulting from the transfer of various l-amino acids or glycine from amino acyl-tRNA to the ɛ-amino group of l-lysine. The UDP-MurNAc-pentapeptide:l-alanine ligase and alanyl-tRNA synthetase genes from Enterococcus faecalis were identified, cloned, and overexpressed in Escherichia coli. The purified enzymes were necessary and sufficient for tRNA-dependent addition of l-alanine to UDP-MurNAc-pentapeptide in vitro. The ligase belonged to the Fem family of proteins, which were initially identified genetically as factors essential for methicillin resistance in Staphylococcus aureus.