Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

Hiroshi M. Sasaki, Shun Ichi Sekine, Toru Sengoku, Ryuya Fukunaga, Motoyuki Hattori, Yukiko Utsunomiya, Chizu Kuroishi, Seiki Kuramitsu, Mikako Shirouzu, Shigeyuki Yokoyama

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-βN) from the archaeon, Pyrococcus horikoshii, at 1.94-Å resolution. PheRS-βN includes the editing domain B3/4, which has archaea eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3 4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe, indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNAPhe deacylation activity. Third, the mutations of archaea eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNAPhe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

Original languageEnglish (US)
Pages (from-to)14744-14749
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number40
StatePublished - Oct 3 2006
Externally publishedYes


  • Aminoacyl-tRNA synthetase
  • Archaea
  • Proofreading
  • X-ray crystallography

ASJC Scopus subject areas

  • General


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