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

Wan, Wei; Tharp, Jeffery M.; Liu, Wenshe Ray

doi:10.1016/j.bbapap.2014.03.002

Cited by 363 publications

(436 citation statements)

References 129 publications

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“…This polyspecificity is also a property of the orthogonal pyrrolysyltRNA synthetase (PylRS)/tRNA Pyl pair (reviewed in ref. 24). PylRS variants that facilitate site-specific insertion of N « -acetyl-L-Lys (AcK; 2) (Fig.…”

mentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

107

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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mentioning

confidence: 99%

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Guo¹,

Wang²,

Nakamura³

et al. 2014

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

107

146

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“…To genetically encode ncAAs in B. cereus, we used the orthogonal pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina barkeri and the amber suppressor tRNA Pyl CUA from Methanosarcina mazei. Variants of this pair have demonstrated high efficiency and fidelity for the incorporation of a wide range of ncAAs in response to the nonsense codons in E. coli (20). Because Bacillus is phylogenetically similar to Escherichia and shares tRNA identity elements with E. coli tRNAs (21), we expected that this tRNA/aaRS pair would similarly be orthogonal (i.e., not cross-react with host tRNAs and aaRSs) in B. cereus.…”

Section: Resultsmentioning

confidence: 99%

Recombinant thiopeptides containing noncanonical amino acids

Luo

Zambaldo

Liu

et al. 2016

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

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Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.noncanonical amino acid | biosynthesis | natural products | thiopeptides | antibiotic

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“…1B) (Wang et al 2006b;Liu and Schultz 2010;Wan et al 2014). To further expand the number and nature of genetically encoded ncAAs, aaRS/tRNA pairs from other archeal and eukaryotic organisms have been recently developed, including Pyrococcus horikoshii lysyl aaRS/tRNA, P. horikoshii glutamyl aaRS/tRNA, Saccharomyces cerevisiae tryptophanyl aaRS/tRNA, heterogeneous leucyl Mt-tRNA/Hs-aaRS, and proly Af-tRNA/Ph-aaRS pairs Santoro et al 2003;Anderson et al 2004;Chatterjee et al 2012Chatterjee et al , 2013dXiao et al 2014).…”

Section: Genetically Encoding Ncaasmentioning

confidence: 99%

“…Using this system, over 200 structurally distinct ncAAs have been genetically encoded in both prokaryotic and eukaryotic organisms. These ncAAs include spectroscopic probes, metal ion chelators, photo-affinity probes and photocaged amino acids, posttranslational modifications, and amino acids with orthogonal chemical activity for the site-specific modification of proteins (Wang et al 2006b;Liu and Schultz 2010;Wan et al 2014). They have been used as probes of protein structure and function, both in vitro and in vivo, and in the rational design of proteins with new or enhanced biological or pharmacological activities.…”

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confidence: 99%

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

Xiao

Schultz

2016

Cold Spring Harb Perspect Biol

115

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The ability to site-specifically incorporate noncanonical amino acids (ncAAs) with novel structures into proteins in living cells affords a powerful tool to investigate and manipulate protein structure and function. More than 200 ncAAs with diverse biological, chemical, and physical properties have been genetically encoded in response to nonsense or frameshift codons in both prokaryotic and eukaryotic organisms with high fidelity and efficiency. In this review, recent advances in the technology and its application to problems in protein biochemistry, cellular biology, and medicine are highlighted.

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

Cited by 363 publications

References 129 publications

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

Recombinant thiopeptides containing noncanonical amino acids

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

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