Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis

Abstract

The FemX _Wv aminoacyl transferase of Weissella viridescens initiates the synthesis of the side chain of peptidoglycan precursors by transferring l -Ala from Ala-tRNA ^Ala to UDP-MurNAc-pentadepsipeptide. FemX _Wv is an attractive target for the development of novel antibiotics, since the side chain is essential for the last cross-linking step of peptidoglycan synthesis. Here, we show that FemX _Wv is highly specific for incorporation of l -Ala in vivo based on extensive analysis of the structure of peptidoglycan. Comparison of various natural and in vitro -transcribed tRNAs indicated that the specificity of FemX _Wv depends mainly upon the sequence of the tRNA although additional specificity determinants may include post-transcriptional modifications and recognition of the esterified amino acid. Site-directed mutagenesis identified cytosines in the G ¹ –C ⁷² and G ² –C ⁷¹ base pairs of the acceptor stem as critical for FemX _Wv activity in agreement with modeling of tRNA ^Ala in the catalytic cavity of the enzyme. In contrast, semi-synthesis of Ala-tRNA ^Ala harboring nucleotide substitutions in the G ³ –U ⁷⁰ wobble base pair showed that this main identity determinant of alanyl-tRNA synthetase is non-essential for FemX _Wv . The different modes of recognition of the acceptor stem indicate that specific inhibition of FemX _Wv could be achieved by targeting the distal portion of tRNA ^Ala for the design of substrate analogues.