Idiosyncratic features in tRNAs participating in bacterial cell wall synthesis.
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 G1-C72 and G2-C71 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 G3-U70 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.
Villet
Régis
R
INSERM, U872, LRMA, Centre de Recherche des Cordeliers, Pôle 4, Equipe 12, Paris, F-75006, France.
Fonvielle
Matthieu
M
Busca
Patricia
P
Chemama
Maryline
M
Maillard
Antoine P
AP
Hugonnet
Jean-Emmanuel
JE
Dubost
Lionel
L
Marie
Arul
A
Josseaume
Nathalie
N
Mesnage
Stéphane
S
Mayer
Claudine
C
Valéry
Jean-Marc
JM
Ethève-Quelquejeu
Mélanie
M
Arthur
Michel
M
D001419:Bacteria / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D001483:Base Sequence; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000378:metabolism; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D012344:RNA, Transfer, Ala / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010457:Peptidoglycan; D012344:RNA, Transfer, Ala; C480726:FemX UDP-MurNAc-pentapeptide l-alanine ligase; D010453:Peptide Synthases