Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase.

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, L-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA(Pyl)). The PylRS-tRNA(Pyl) pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N(epsilon)-(tert-butoxycarbonyl)-L-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the alpha-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain alpha-groups: alpha-hydroxyacid, non-alpha-amino-carboxylic acid, N(alpha)-methyl-amino acid, and D-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA(Pyl) in E. coli in the presence of the alpha-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA(Pyl) pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.