title: 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.
authors.affiliation: Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
authors.forename: Hiroshi M
authors.initials: HM
authors.lastname: Sasaki
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Sekine
authors.forename: Toru
authors.initials: T
authors.lastname: Sengoku
authors.forename: Ryuya
authors.initials: R
authors.lastname: Fukunaga
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Hattori
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Utsunomiya
authors.forename: Chizu
authors.initials: C
authors.lastname: Kuroishi
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D050505:Mutant Proteins; D021122:Protein Subunits; D012346:RNA, Transfer, Amino Acyl; D014443:Tyrosine; D010652:Phenylalanine-tRNA Ligase