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

Abstract：

: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 beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) 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-tRNA(Phe), 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-tRNA(Phe) 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-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Sasaki HM,Sekine S,Sengoku T,Fukunaga R,Hattori M,Utsunomiya Y,Kuroishi C,Kuramitsu S,Shirouzu M,Yokoyama S

doi

10.1073/pnas.0603182103

subject

Has Abstract

pub_date

2006-10-03 00:00:00

pages

14744-9

issue

eissn

0027-8424

issn

1091-6490

pii

0603182103

journal_volume

103

pub_type

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