Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis.
Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemX(Wv). Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemX(Wv) with the acceptor arm of tRNA(Gly) and with L-Ser or larger residues quantitatively accounts for the preferential transfer of L-Ala observed with complete aminoacyl-tRNAs. The main FemX(Wv) identity determinant was identified as the penultimate base pair (G(2)-C(71)) of the acceptor arm instead of G(3)*U(70) for the alanyl-tRNA synthetase. FemX(Wv) tolerated a configuration inversion of the Calpha of L-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemX(Wv) is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNA(Gly) from the active site.
Fonvielle
Matthieu
M
Centre de Recherche des Cordeliers, LRMA, Equipe 12, INSERM, Université Pierre et Marie Curie - Paris 6, France.
Chemama
Maryline
M
Villet
Régis
R
Lecerf
Maxime
M
Bouhss
Ahmed
A
Valéry
Jean-Marc
JM
Ethève-Quelquejeu
Mélanie
M
Arthur
Michel
M
D000409:Alanine / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D019884:Nitrogenous Group Transferases / Q000378:metabolism*; D012344:RNA, Transfer, Ala / Q000737:chemistry / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D012344:RNA, Transfer, Ala; D012346:RNA, Transfer, Amino Acyl; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C002225:UDP-N-acetylmuramic acid pentapeptide; D012694:Serine; D019884:Nitrogenous Group Transferases; C476453:UDP-N-acetylmuramoyl pentapeptide-lysine N(6)-alanyltransferase; D000409:Alanine; D005998:Glycine