Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation

Abstract

The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 Å resolution. The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs. In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes. Its structure explains the unique property of archaeal AspRSs of accommodating both tRNA Asp and tRNA Asn . Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed. Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal. Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes. The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation. An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made. This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes.

Evolutionary Divergence of the Archaeal Aspartyl-tRNA Synthetases into Discriminating and Nondiscriminating Forms

“…18). We suggest that a discriminating AspRS is common to the pyrococcal group, despite the attractive observation that misacylation may be achieved by a shortened loop identified in the P. kodakaraensis AspRS structure (10), which corresponds to a loop in the yeast AspRS structure that interacts with the C36 in the tRNA Asp anticodon (41). Position 36 is possibly a major discriminating element because it is the only anticodon position to differ between tRNA Asp and tRNA Asn .…”

“…The fact that two discriminating forms are now found in both the bacteria-type (17) and archaea-type AspRSs may not be surprising upon comparison of their N-terminal domains, which are responsible for the specific recognition of the tRNA tRNA Specificity of Archaeal Aspartyl-tRNA Synthetasesanticodon loop. These ϳ130-residue domains are closer in sequence between the bacterial and the archaeal AspRSs than either is with the eukaryotic AspRS (10,12), which is still a presumably discriminating AspRS. No eukaryon is known to lack AsnRS in the cytoplasm, and no mischarging eukaryal AspRS has yet been described.…”

“…The phylogenetic segregation of the AspRSs into two groups is also supported by comparisons of AspRS structures from the three domains (8 -11). Several residues critical for binding of the Asp-AMP adenylate are unique to the eukaryote-and archaea-type AspRSs and are distinct from those used in the bacteria-type enzyme (10). Comparison of AspRS amino acid sequences reveals a relatively long (ϳ570 residue) enzyme common in the bacteria, compared with a shorter (ϳ430 residue) enzyme in the archaea.…”

“…Biochemical analyses led to the proposal that the bacterial AspRS was a strictly discriminating enzyme, whereas the archaeal AspRS could aspartylate tRNA Asp and tRNA Asn (10,13,14). Both Thermus thermophilus and Deinococcus radiodurans were shown to contain a discriminating bacteria-type AspRS, which charged only tRNA Asp , and a nondiscriminating ar-chaea-type AspRS, which also misacylated tRNA Asn (14 -16).…”

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change

“…The archaeal genre comprises all archaeal AspRS enzymes, as well as one of two types of AspRSs found in the bacteria Thermus thermophilus (12), Deinococcus radiodurans (13), and Clostridium acetobutylicum. Whereas the archaeal AspRS enzymes were thought to be nondiscriminating (12,14), it is now known that in several archaea (including Thermococcus kodakaraensis), this enzyme is discriminating (15). In contrast to bacterial AspRS proteins, the tRNA specificity of the archaeal enzyme can be predicted from the whole genome content (15): a ND-AspRS always exists in the genome together with the archaeal Asp-tRNA Asn amidotransferase, whereas a D-AspRS accompanies an asparaginyl-tRNA synthetase (AsnRS).…”