Pyrrolysyl‐tRNA synthetase variants reveal ancestral aminoacylation function

Ko, Jin Hong; Wang, Yane‐Shih; Nakamura, Akiyoshi; Guo, Long; Söll, Dieter; Umehara, Takuya

doi:10.1016/j.febslet.2013.08.018

Cited by 31 publications

(39 citation statements)

References 22 publications

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“…We determined the substrate range of two AcKRS enzymes by screening with a chemically diverse library of 313 ncAAs (11,32), followed by rescreening with a selected subset of Phe and Lys derivatives (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

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108

146

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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%

“…1B). For this screen, we used a superfolder GFP (sfGFP) reporter (32) bearing a TAG codon at position 2, a site known to be permissive for diverse ncAAs (32,33). The reporter was coexpressed with plasmids encoding the tRNA Pyl and AcKRS; quantification was by sfGFP fluorescence, the indication of ncAA incorporation efficiency.…”

Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

Self Cite

108

146

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“…After a PylRS library with randomization at six active site residues was selected for amber suppression ability in E. coli in LB medium, multiple clones responsive to Phe were identified. [115, 116] This result opened a new avenue of the PylRS research area which is to develop PylRS mutants for the genetic incorporation of Phe derivatives. Although a number of Phe derivatives have been genetically encoded in bacteria and eukaryotes using engineered tyrosyl-tRNA synthase-tRNA Pyl pairs that were originally from Methanocaldococcus jannaschii and E. coli ,[3, 117–119] the relatively relaxed substrate binding makes the engineering of PylRS more straightforward and the high orthogonally of the native Pyl system in different cell strains also allows the more convenient transfer of engineering Pyl systems between different cells.…”

Section: An Outstanding Genetic Code Expansion Toolmentioning

confidence: 99%

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Wan¹,

Tharp²,

Liu³

2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

363

418

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The genetic incorporation of the 22nd proteinogenic amino acid, pyrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNAPyl. Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNAPyl. These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

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“…Thus, canonical AA mischarging by o-aaRS variants [69] and suppressor tRNAs that are not fully orthogonal obscure results. The latter may abolish the possibility of negative selection [33 •• ].…”

Section: Figurementioning

confidence: 99%

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

Vargas-Rodriguez

Sevostyanova

Söll

et al. 2018

Current Opinion in Chemical Biology

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107

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Synthesis of proteins with non-canonical amino acids via genetic code expansion is at the forefront of synthetic biology. Progress in this field has enabled site-specific incorporation of over 200 chemically and structurally diverse amino acids into proteins in an increasing number of organisms. This has been facilitated by our ability to repurpose aminoacyl-tRNA synthetases to attach non-canonical amino acids to engineered tRNAs. Current efforts in the field focus on overcoming existing limitations to the simultaneous incorporation of multiple non-canonical amino acids or amino acids that differ from the l-α-amino acid structure (e.g. d-amino acid or β-amino acid). Here, we summarize the progress and challenges in developing more selective and efficient aminoacyl-tRNA synthetases for genetic code expansion.

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Pyrrolysyl‐tRNA synthetase variants reveal ancestral aminoacylation function

Cited by 31 publications

References 22 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

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