Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase

@article{Polycarpo2006PyrrolysineAA,
  title={Pyrrolysine analogues as substrates for pyrrolysyl‐tRNA synthetase},
  author={C. Polycarpo and S. Herring and A. B{\'e}rub{\'e} and J. L. Wood and D. S{\"o}ll and A. Ambrogelly},
  journal={FEBS Letters},
  year={2006},
  volume={580}
}
In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in‐frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNAPyl by pyrrolysyl‐tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNAPyl. We report here the in vitro aminoacylation of tRNAPyl by PylRS with two Pyl analogues: N‐ε‐d‐prolyl‐l‐lysine (d‐prolyl‐lysine… Expand
The amino‐terminal domain of pyrrolysyl‐tRNA synthetase is dispensable in vitro but required for in vivo activity
TLDR
It is shown that the amino‐terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for Pyl RS function in vivo. Expand
Pyrrolysyl-tRNA synthetase:tRNAPyl structure reveals the molecular basis of orthogonality
TLDR
It is shown that Desulfitobacterium hafniense PylRS–tRNAPyl is an orthogonal pair in vitro and in vivo, and the crystal structure of this orthogonality pair is presented. Expand
Specificity of pyrrolysyl-tRNA synthetase for pyrrolysine and pyrrolysine analogs.
TLDR
Interactions necessary for recognition of substrates by archaeal PylS are probed via synthesis of close pyrrolysine analogs and testing their reactivity in amino acid activation assays, illustrating the relative importance of the H-bonding and hydrophobic interactions in the recognition of the methylpyrroline ring of pyr rolysine. Expand
Evolving the N‐Terminal Domain of Pyrrolysyl‐tRNA Synthetase for Improved Incorporation of Noncanonical Amino Acids
TLDR
By evolving the N‐terminal domain of PylRS that directly interacts with tRNAPyl, a mutant clone displaying improved amber‐suppression efficiency for the genetic incorporation of Nϵ‐(tert‐butoxycarbonyl)‐l‐lysine threefold more than the wild type was identified. Expand
Recognition of non-alpha-amino substrates by pyrrolysyl-tRNA synthetase.
TLDR
It is reported here that wild type PylRS can recognize substrates with a variety of main-chain alpha-groups: alpha-hydroxyacid, non-alpha-amino-carboxylic acid, N(alpha)-methyl-amINO acid, and D-aminos acid, each with the same side chain as that of Boc-lysine, in contrast, PheRS recognizes none of these amino acid analogs. Expand
Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation
TLDR
Three crystal structures of the Methanosarcina mazei PylRS complexed with either AMP–PNP, Pyl–AMP plus pyrophosphate, or the Pyl analogue N-ε-[(cylopentyloxy)carbonyl]-l-lysine plus ATP are determined, revealing that Pyrrolysine becomes attached to its cognate tRNA by pyrrolysyl-tRNA synthetase (PylRS). Expand
Misacylation of pyrrolysine tRNA in vitro and in vivo
TLDR
It is shown that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkingeri bacterial‐type seryl‐tRNA synthetase in vitro and in vivo in Escherichia coli. Expand
The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine
TLDR
The results indicate that the radical S-adenosyl-l-methionine (SAM) protein PylB mediates a lysine mutase reaction that produces 3-methylornithine, which is then ligated to a second molecule of l Lysine by PylC before oxidation byPylD results in pyrrolysine. Expand
Translation of UAG as Pyrrolysine
TLDR
Pyrrolysine incorporation appears to occur to some extent by amber suppression on a genome-wide basis in methanogenic Archaea, and some methanogen genomes encode additional homologs of elongation and release factors, however, their limited distribution suggests at best a nonessential role in enhancing UAG translation as pyrrolesine. Expand
Adding pyrrolysine to the Escherichia coli genetic code
TLDR
The impact of the presence of PYLIS on the level of Pyl and the Pyl analog N‐ε‐cyclopentyloxycarbonyl‐l‐lysine (Cyc) incorporation using a quantitative lacZ–luc tandem reporter system in an Escherichia coli context is studied. Expand
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