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Abstract The crystal structures of histidyl- (HisRS) and threonyl-tRNA synthetase (ThrRS) from E. coli and glycyl-tRNA synthetase (GlyRS) from T. thermophilus, all homodimeric class IIa enzymes, were determined in enzyme-substrate and enzyme-product states corresponding to the two steps of aminoacylation. HisRS was complexed with the histidine analog histidinol plus ATP and with histidyl-adenylate, while GlyRS was complexed with ATP and with glycyl-adenylate; these complexes represent the enzyme-substrate and enzyme-product states of the first step of aminoacylation, i.e. the amino acid activation. In both enzymes the ligands occupy the substrate-binding pocket of the N-terminal active site domain, which contains the classical class II aminoacyl-tRNA synthetase fold. HisRS interacts in the same fashion with the histidine, adenosine and α-phosphate moieties of the substrates and intermediate, and GlyRS interacts in the same way with the adenosine and α-phosphate moieties in both states. In addition to the amino acid recognition, there is one key mechanistic difference between the two enzymes: HisRS uses an arginine whereas GlyRS employs a magnesium ion to catalyze the activation of the amino acid. ThrRS was complexed with its cognate tRNA and ATP, which represents the enzyme-substrate state of the second step of aminoacylation, i.e. the transfer of the amino acid to the 3'-terminal ribose of the tRNA. All three enzymes utilize class II conserved residues to interact with the adenosine-phosphate. ThrRS binds tRNA(Thr) so that the acceptor stem enters the active site pocket above the adenylate, with the 3'-terminal OH positioned to pick up the amino acid, and the anticodon loop interacts with the C-terminal domain whose fold is shared by all three enzymes. We can thus extend the principles of tRNA binding to the other two enzymes.

Aminoacyl-tRNA synthetases (aaRS) bind their substrates-ATP, amino acids and tRNA- and stabilize putative transition states in the aminoacylation reaction. Here, we discuss the common and distinguishing structural and functional themes of the 20 known aaRS, which can be divided into two main classes (I and II) and into further subgroups on this basis.