Abstract:The genetic code in a eukaryotic system has been expanded by the engineering of
Escherichia coli
tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo-
l
-tyrosine rather than
l
-tyrosine. In the present study, we determined the 3-iodo-
l
-tyrosine- and
l
-tyrosine-bound structures of the 37V195C mutant of the
E. coli
… Show more
“…We used a reported crystal structure for the MjTyrRS 36 to inform the diversification of 12 residues in the amino acid binding pocket surrounding the variable side chain of the nsAA (compared with typically six or fewer residues 18,37,38 , with few exceptions targeting nine residues 39 ), and five residues at the aaRS-tRNA CUA anticodon recognition interface (Fig. 3a).…”
Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (~50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.
“…We used a reported crystal structure for the MjTyrRS 36 to inform the diversification of 12 residues in the amino acid binding pocket surrounding the variable side chain of the nsAA (compared with typically six or fewer residues 18,37,38 , with few exceptions targeting nine residues 39 ), and five residues at the aaRS-tRNA CUA anticodon recognition interface (Fig. 3a).…”
Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (~50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.
“…By contrast, in the reported structure, iodoTyrRS- ec recognizes 3-iodo- l -tyrosine by making van der Waals contacts between the mutated residues, Val37 and Cys195, and the iodine atom (Figure 4B). The binding of l -tyrosine to iodoTyrRS- ec , on the other hand, is weakened by the Tyr37Val substitution, which removes the hydrogen bond between the phenolic hydroxyl groups of l -tyrosine and Tyr37 (28) (Figure 4A). Figure 4.( A ) The amino-acid binding pocket in the crystal structure of iodoTyrRS- ec bound with 3-azido- l -tyrosine.…”
Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion.
“…Previous structural studies of aminoacyl-tRNA synthetases that recognize unnatural amino acids have shown little change in the backbone structures compared with the WT structure. For example, both the substrate-bound and Tyr-bound structures of a mutant E. coli tyrosyl synthetase that recognizes 3-iodo-L-Tyr have very similar backbone traces as the WT synthetase bound to Tyr (27). The backbone structure of tryptophanyl tRNA synthetase from Deinococcus radiodurans bound to 5-hydroxy-Trp (5HT) is similar to the Trp-bound structure, although the orientation of 5HT in the active site is rotated by almost 180°compared with Trp (28).…”
Section: Comparison With Other Mj Tyrrs Structuresmentioning
Recently, tRNA aminoacyl-tRNA synthetase pairs have been evolved that allow one to genetically encode a large array of unnatural amino acids in both prokaryotic and eukaryotic organisms. We have determined the crystal structures of two substrate-bound Methanococcus jannaschii tyrosyl aminoacyl-tRNA synthetases that charge the unnatural amino acids p-bromophenylalanine and 3-(2-naphthyl)alanine (NpAla). A comparison of these structures with the substratebound WT synthetase, as well as a mutant synthetase that charges p-acetylphenylalanine, shows that altered specificity is due to both side-chain and backbone rearrangements within the active site that modify hydrogen bonds and packing interactions with substrate, as well as disrupt the ␣8-helix, which spans the WT active site. The high degree of structural plasticity that is observed in these aminoacyltRNA synthetases is rarely found in other mutant enzymes with altered specificities and provides an explanation for the surprising adaptability of the genetic code to novel amino acids.x-ray crystal structure ͉ unnatural amino acids ͉ expanded genetic code ͉ molecular evolution W ith the rare exceptions of selenocysteine (1) and pyrrolysine (2, 3), the common 20 amino acids are conserved across all known organisms. However, there does not appear to be an inherent limit to the size or chemical nature of the genetic code, since it has been shown that additional amino acids can be genetically encoded in both prokaryotic and eukaryotic organisms in response to nonsense or frameshift codons (4, 5). This requires a unique codon-suppressor tRNA pair and the corresponding aminoacyl-tRNA synthetase, which do not crossreact with the amino acids, tRNAs, or synthetases of the host organism (4, 5). The specificity of the aminoacyl tRNA synthetase is then altered by generating large libraries of active-site mutants and passing them through positive and negative selections to identify synthetases that selectively acylate the cognate tRNA with the unnatural amino acid but not any of the common amino acids. This approach has been used to add Ͼ30 unnatural amino acids to the genetic codes of bacteria, yeast, and mammalian cells with high fidelity and efficiencies.In Escherichia coli, a tyrosyl aminoacyl-tRNA synthetase (TyrRS) tRNA CUA Tyr pair from the archea Methanococcus jannaschii (Mj) was used as an orthogonal tRNA synthetase pair (6). Mutant synthetases have been evolved that selectively aminoacylate their cognate suppressor tRNAs with glycosylated (7), photoreactive (8), and metal-binding amino acids, as well as amino acids with unique functional groups (9-11) . To establish the molecular basis for the surprising adaptability of this synthetase, we solved (12) the structure of a mutant Mj TyrRS that is selective for p-acetylphenylalanine (p-AcPhe). The x-ray crystal structure revealed significant structural changes within the enzyme active site that result from the mutations Y32L, D158G, I159C, and L162R. The Y32L and D158G mutations remove two hydrogen bonds (H-bonds) with the...
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