Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Journal Article · Tue Aug 17 04:00:00 EDT 2010 · J. Mol. Biol.

DOI:https://doi.org/10.1016/j.jmb.2009.06.014· OSTI ID:1006132

Wu, Jing; Bu, Weishu; Sheppard, Kelly; Kitabatake, Makoto; Kwon, Suk-Tae; Söll, Dieter; Smith, Janet L

Many bacteria form Gln-tRNA{sup Gln} and Asn-tRNA{sup ASN} by conversion of the misacylated Glu-tRNA{sup Gln} and Asp-tRNA{sup ASN} species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction. A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn{sup 2+} site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn{sup 2+} binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA{sup Gln} or Asn-tRNA{sup ASN}.

• Research Organization: Argonne National Laboratory (ANL)
• Sponsoring Organization: USDOE
• OSTI ID: 1006132
• Journal Information: J. Mol. Biol., Journal Name: J. Mol. Biol. Journal Issue: (4) ; 08, 2009 Vol. 391; ISSN JMOBAK; ISSN 0022-2836
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

36 MATERIALS SCIENCE
AMIDES
AMINO ACIDS
AMMONIA
ASPARAGINE
BACTERIA
CATALYSIS
CRYSTAL STRUCTURE
ENZYMES
GLUTAMINE
LIGASES
STAPHYLOCOCCUS
SUBSTRATES