Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient <i>in Vivo</i> Incorporation of Structurally Divergent Non-native Amino Acids

Takimoto, Jeffrey K.; Dellas, Nikki; Noel, Joseph P.; Wang, Lei

doi:10.1021/cb200057a

Cited by 95 publications

(118 citation statements)

References 40 publications

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“…Structural analyses have also revealed how mutant PylRS enzymes can accommodate ncAAs that are chemically distinct (50) and larger than Pyl (47), including furan-containing Lys (46) and norbornene-containing Pyl analogs (51). Larger ncAAs are accepted by these PylRS variants by creating larger amino acid recognition pockets with mutations to smaller active site residues, particularly at position 306.…”

Section: Resultsmentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

104

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%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

104

146

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“…[124] Wang et al showed that PylRS mutants selective for 56 , 96 , and 97 could be evolved. [125, 126]…”

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

350

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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“…However, the Pyl-tRNA is not hardwired for amber codon suppression, and can be adapted to decode a number of other codons (Ambrogelly et al, 2007). In addition, the PylRS has been shown to readily accept a variety of side chain structures (Polycarpo et al, 2006;Yanagisawa et al, 2008;Neumann et al, 2008;Chen et al, 2009;Nguyen et al, 2009;Li et al, 2009;Hancock et al, 2010;Ou et al, 2011;Plass et al, 2011;Takimoto et al, 2011) as well as a set of non-alpha amino derivatives (Kobayashi et al, 2009). This feature makes the PylRS/tRNA pair an ideal candidate for site specific integration of NNAAs.…”

Section: Methods For Evolution Of Aarss and Trnas For Nnaasmentioning

confidence: 99%