<i>N</i>‐Acetyl lysyl‐tRNA synthetases evolved by a CcdB‐based selection possess <i>N</i>‐acetyl lysine specificity in vitro and in vivo

Umehara, Takuya; Kim, Jihyo; Lee, Sangsik; Guo, Long; Söll, Dieter; Park, Hee-Sung

doi:10.1016/j.febslet.2012.01.029

Cited by 84 publications

(111 citation statements)

References 27 publications

(47 reference statements)

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“…Seven distinct AckRS variants have been reported previously (25)(26)(27)(28)30). Three variants were derived from the MbPylRS (25,30), whereas the others originated from the MmPylRS (26)(27)(28).…”

Section: Resultsmentioning

confidence: 99%

“…Three variants were derived from the MbPylRS (25,30), whereas the others originated from the MmPylRS (26)(27)(28). Biochemical studies revealed that the AcKRS enzymes were far less catalytically efficient than natural tRNA synthetases (26). This result led us to characterize AcKRS complexed with AcK structurally.…”

Section: Resultsmentioning

confidence: 99%

“…The AcKRS3 structure indicates that the Leu309Ala substitution creates space for a new cluster of hydrophobic and bulky residues at positions 348 and 306. These mutations lead to an AcKRS active site that is smaller and more hydrophobic than that of the parent PylRS, which demonstrates why AcKRS is no longer active with the larger substrate, Pyl (26) ( Table 2) and why the parent PylRS enzyme is not active in inserting AcK into proteins in vivo (26). Interestingly, one PylRS variant (Tyr306Ala/Tyr384Phe) was shown to accommodate furan-containing ncAAs that are substantially larger than Pyl (46).…”

Section: Resultsmentioning

confidence: 99%

“…PylRS variants that facilitate site-specific insertion of N « -acetyl-L-Lys (AcK; 2) (Fig. 1A) into proteins were derived from directed evolution experiments (25)(26)(27)(28). These AcK-tRNA synthetase (AcKRS) enzymes have been used to investigate the role of acetylation sites in tumor suppressor p53 (29) and histone H3 (30).…”

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confidence: 99%

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Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA Pyl have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N e -acetyl-Lys (AcK) onto tRNA Pyl . Here, we examine an N e -acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids.aminoacyl-tRNA synthetase | genetic code | genetic selection | posttranslational modification | synthetic biology T he standard genetic code table relates the 64 nucleotide triplets to three stop signals and 20 canonical amino acids. Some organisms, including humans, naturally evolved expanded genetic codes that accommodate 21 amino acids (1), or possibly 22 amino acids in rare cases (2). Engineering translation system components, including tRNAs (3, 4), aminoacyl-tRNA synthetases (AARSs) (5, 6), elongation factors (7), and the ribosome itself (8), have produced organisms with artificially expanded genetic codes. Products of genetic code engineering include bacterial, yeast, and mammalian cells and animals that are able to synthesize proteins with sitespecifically inserted noncanonical amino acids (ncAAs) (9).Genetic code expansion systems rely on an orthogonal AARS/ tRNA pair (o-AARS, o-tRNA) (5, 6). The o-AARS should be specific in ligating a desired ncAA to a stop codon decoding tRNA, and both the o-tRNA and o-AARS are assumed not to cross-react with endogenous AARSs or tRNAs. Although some AARSs evolved in nature to recognize certain ncAAs (10-12), many genetic code expansion systems require a mutated AARS active site. The active site of the o-AARS is usually redesigned via directed evolution (6), including positive and negative selective rounds, to produce an enzyme that is assumed to be specific for an ncAA and not active with the 20 canonical amino acids. Genetic code expansion technology is rapidly evolving (13), and the ability to incorporate multiple ncAAs into a protein using quadruplet-codon decoding (14) or sense-codon recoding (15-19) is now becoming feasible. Protein synthesis with multiple ncAAs will require o-AARSs that are able to discriminate their ncAA substrate not only from canonical amino acids in the cell but from other ncAAs that are added to the cell.Probing the effects of amino acid analogs on bacterial cell...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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104

146

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“…Typically, a UAG stop codon is assigned to the nsAA, and the orthogonal tRNA anticodon is mutated to CUA. Pairs of aaRSs and tRNAs from organisms such as Methanocaldococcus jannaschii and Methanosarcina species have been used to incorporate a wide variety of nsAAs 4,17 into proteins in bacterial hosts 4,18,19 , but at only one or a few instances within a polypeptide chain 5,10 .…”

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confidence: 99%

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

et al. 2015

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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.

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Genetically encoding thioacetyl‐lysine as a non‐deacetylatable analog of lysine acetylation in Escherichia coli

et al. 2017

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Reversible lysine acetylation is one of the most widely distributed post‐translational modifications; it is involved in a variety of biological processes and can be found in all three domains of life. Acetyltransferases and deacetylases work coordinately to control levels of protein acetylation. In this work, we applied the genetic code expansion strategy to site‐specifically incorporate Nε‐thioacetyl‐l‐lysine (TAcK) as an analog of Nε‐acetyl‐l‐lysine (AcK) into green fluorescent protein and malate dehydrogenase in Escherichia coli. We showed that TAcK could serve as an ideal functional mimic for AcK. It could also resist the bacterial sirtuin‐type deacetylase CobB. Thus, genetic incorporation of TAcK as a non‐deacetylatable analog of AcK into proteins will facilitate in vivo studies of protein acetylation.

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N‐Acetyl lysyl‐tRNA synthetases evolved by a CcdB‐based selection possess N‐acetyl lysine specificity in vitro and in vivo

Cited by 84 publications

References 27 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Genetically encoding thioacetyl‐lysine as a non‐deacetylatable analog of lysine acetylation in Escherichia coli

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