title: Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography.
abstract: We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Murayama
authors.forename: Kenji
authors.initials: K
authors.lastname: Oki
authors.forename: Fumie
authors.initials: F
authors.lastname: Iraha
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kato-Murayama
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takahashi
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Ohtake
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kobayashi
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: D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D017016:Methanococcaceae / Q000201:enzymology / Q000378:metabolism; D007470:Monoiodotyrosine / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: C523964:3-iodotyrosine; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C521051:RimL protein, E coli; D001191:Arylamine N-Acetyltransferase; D007470:Monoiodotyrosine