Crystallographic Studies on Multiple Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase

Yanagisawa, Takatoshi; Ishii, Ryohei; Fukunaga, Ryuya; Kobayashi, T.; Sakamoto, Kensaku; Yokoyama, Shigeyuki

doi:10.1016/j.jmb.2008.02.045

Cited by 91 publications

(172 citation statements)

References 56 publications

(108 reference statements)

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“…The enzyme kinetic constants (k cat and K m ) are used to compare activity for the different ncAAs. As reported in earlier studies (38,39), the truncated version (to overcome solubility problems) of the PylRS enzyme (residues 185-454) was used to measure the ATP-PP i exchange. However, in the aminoacylation experiments, full-length PylRS variants could be used because the assay (37) required low enzyme concentrations.…”

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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“…Moreover, pyrrolysine turned out to be relatively unstable, which precluded its storage for ex- tended periods of time. After being unable, in contrast to others, [12,15] to reproduce our synthetic protocol for the parent compound 1, Ambrogelly et al [16] reported on the synthesis of the pyrrolysine isomer 2 ( Figure 3) but were also dissatisfied with the shortcomings of their approach. To alleviate these problems, a series of lysine derivatives 2-8 ( Figure 3) was screened for their ability to serve as pyrrolysine surrogates using an aminoacylation assay based on acid-urea gel electrophoresis and Northern blotting.…”

Section: Exploratory Search For Pyrrolysine Analogsmentioning

confidence: 87%

“…[12] Building on this observation, Yokoyama et al identified a series of pyrrolysine analogs 19-21 and ent-14 ( Figure 6) that were shown to be efficiently ligated to tRNA Pyl by M. mazei PylRS. [20] Moreover, the hydroxy acid 20 was incorporated into a protein in vivo, thus enabling the site-specific introduction of an ester bond.…”

Section: Exploratory Search For Pyrrolysine Analogsmentioning

confidence: 99%

“…[11] From the results of these studies, it was concluded that the highly specialized and intricate interaction surface between the two units, which is markedly distinct from those observed for other known aaRStRNA complexes, is the key feature responsible for the PylRS-tRNA Pyl orthogonality. Moreover, crystallographic analyses of the M. mazei [12,13] and D. hafniense [14] PylRS have revealed a remarkably large binding pocket for 1 that, in turn, suggests broad specificity for lysine analogs bearing a wide variety of N-6 side-chains.…”

Section: Introductionmentioning

confidence: 99%

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Pyrrolysine Analogs for Translational Incorporation into Proteins

Fekner

Chan

2010

Eur J Org Chem

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The discovery that pyrrolysine is translationally incorporated into methylamine methyltransferases from certain methanogens in response to an in‐frame amber codon prompted detailed studies of the biochemical machinery associated with its genetic encoding. Over the past few years, significant progress has also been made in identifying a set of structural and electronic features that enable a compound to act as an effective pyrrolysine mimic. With such empirical knowledge in hand, it is becoming increasingly feasible to rationally design and synthesize new analogs with useful chemical, physical, or biochemical properties and then site‐specifically incorporate them into proteins. The present microreview highlights recent key developments in this rapidly expanding area of research.

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“…13 The recombinant functionality of PylS and tRNA Pyl have highlighted their potential as an orthologous pair in E. coli 10 that can be used for production of recombinant proteins with new biochemical properties due to uniquely inserted residues under the direction of an amber codon. 14 Although the synthesis of stereochemically pure pyrrolysine has been reproducibly achieved, 6,15 the difficulty of this synthesis has led to the use of analogs of pyrrolysine. One such compound is enPyl.…”

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confidence: 99%