2zin

Publication Abstract from PubMed

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.

Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.,Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S Chem Biol. 2008 Nov 24;15(11):1187-97. PMID:19022179^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.