title: Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.
abstract: Pyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNA(Pyl). In this study, N(epsilon)-(tert-butyloxycarbonyl)-L-lysine (BocLys) and N(epsilon)-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNA(Pyl) by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an N(epsilon)-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for N(epsilon)-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing N(epsilon)-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.
authors.affiliation: Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yanagisawa
authors.forename: Ryohei
authors.initials: R
authors.lastname: Ishii
authors.forename: Ryuya
authors.initials: R
authors.lastname: Fukunaga
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D049148:Aminoacylation; D001386:Azides; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004951:Esterification; D005456:Fluorescent Dyes / Q000378:metabolism; D016153:Genes, Suppressor; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017020:Methanosarcina / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D012343:RNA, Transfer / Q000378:metabolism
chemical_list: D001386:Azides; D005456:Fluorescent Dyes; D050505:Mutant Proteins; C534884:N(epsilon)-(2-azidobenzyloxycarbonyl)lysine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D008250:Lysine-tRNA Ligase; C456839:pyrrolysine; D008239:Lysine