Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1391650 | Chemistry & Biology | 2008 | 11 Pages |
SummaryPyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nɛ-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nɛ-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl 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 Nɛ-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 Nɛ-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 Nɛ-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation.