Aminoacylation at the atomic level in class IIa aminoacyl-tRNA synthetases

The crystal structures of histidyl- (HisRS) and threonyl-tRNA synthetase (T hrRS) from E. coil and glycyl-tRNA synthetase (GlyRS) from T. thermophilus, all homodimeric class Ila enzymes, were determined in enzyme-substrate and enzyme-product states corresponding to the two steps of aminoacylation. Hi sRS was complexed with the histidine analog histidinol plus ATP and with hi stidyl-adenylate, while GlyRS was complexed with ATP and with glycyl-adenyl ate; these complexes represent the enzyme-substrate and enzyme-product stat es 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-termi nal active site domain, which contains the classical class II aminoacyl-tRN A synthetase fold. HisRS interacts in the same fashion with the histidine, adenosine and alpha -phosphate moieties of the substrates and intermediate, and GlyRS interacts in the same way with the adenosine and alpha -phosphat e moieties in both states. In addition to the amino acid recognition, there is one key mechanistic difference between the two enzymes: HisRS uses an a rginine whereas GlyRS employs a magnesium ion to catalyze the activation of the amino acid. ThrRS was complexed with its cognate tRNA and ATP, which r epresents 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 positi oned to pick up the amino acid, and the anticodon loop interacts with the C -terminai domain whose fold is shared by all three enzymes. We can thus ext end the principles of tRNA binding to the other two enzymes.