Mechanism of a GatCAB amidotransferase: aspartyl-tRNA synthetase increases its affinity for Asp-tRNA(Asn) and novel aminoacyl-tRNA analogues are competitive inhibitors.
The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.
Huot
Jonathan L
JL
Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines, Département de Biochimie et de Microbiologie, et Département de Chimie, Université Laval, Québec, Québec, Canada G1K 7P4.
Balg
Christian
C
Jahn
Dieter
D
Moser
Jürgen
J
Emond
Audrey
A
Blais
Sébastien P
SP
Chênevert
Robert
R
Lapointe
Jacques
J
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D019881:Aminoacyltransferases / Q000378:metabolism*; D000617:Aminoglycosides / Q000378:metabolism; D001226:Aspartate-tRNA Ligase / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology; D019884:Nitrogenous Group Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000617:Aminoglycosides; C525151:aspartycin; C444059:glutamycin; D001224:Aspartic Acid; D019881:Aminoacyltransferases; C023175:transamidases; C412016:Asp-tRNA(Asn) amidotransferase; C525150:GatCAB amidotransferase, Helicobacter pylori; D019884:Nitrogenous Group Transferases; D001226:Aspartate-tRNA Ligase; D000241:Adenosine