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

JOURNAL OF MOLECULAR BIOLOGY

卷:391

Article

Wu, Jing¹  Bu, Weishu¹  Sheppard, Kelly²  Kitabatake, Makoto³  Kwon, Suk-Tae²  Soell, Dieter^2,4  Smith, Janet L.¹ 

[1] Univ Michigan, Inst Life Sci, Dept Biol Chem, Ann Arbor, MI 48109 USA

[2] Yale Univ, Dept Mol Biophys & Biochem, New Haven, CT 06520 USA

[3] Kyoto Univ, Inst Virus Res, Dept Genet & Mol Biol, Kyoto 6068507, Japan

[4] Yale Univ, Dept Chem, New Haven, CT 06520 USA

关键词: tRNA-dependent amidotransferase;    GatCAB;    crystal structure;    amidase superfamily;    amidotransferase evolution;   

DOI  :  10.1016/j.jmb.2009.06.014

来源: Elsevier

【 摘 要 】

Many bacteria form Gln-tRNA(Gln) and Asn-tRNA(Asn) by conversion of the misacylated Glu-tRNA(Gln) and Asp-tRNA(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 Zn2+ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn2+ 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(Gln) or Asn-tRNA(Asn) (C) 2009 Elsevier Ltd. All rights reserved.

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