Pyrrolysine Explained

Pyrrolysine should not be confused with Pyrolysis.

Pyrrolysine (symbol Pyl or O;[1] encoded by the 'amber' stop codon UAG) is an α-amino acid that is used in the biosynthesis of proteins in some methanogenic archaea and bacteria;[2] [3] it is not present in humans. It contains an α-amino group (which is in the protonated – form under biological conditions) and a carboxylic acid group (which is in the deprotonated –COO− form under biological conditions). Its pyrroline side-chain is similar to that of lysine in being basic and positively charged at neutral pH.

Genetics

Nearly all genes are translated using only 20 standard amino acid building blocks. Two unusual genetically-encoded amino acids are selenocysteine and pyrrolysine. Pyrrolysine was discovered in 2002 at the active site of methyltransferase enzyme from a methane-producing archeon, Methanosarcina barkeri.[4] This amino acid is encoded by UAG (normally a stop codon), and its synthesis and incorporation into protein is mediated via the biological machinery encoded by the pylTSBCD cluster of genes.

Composition

As determined by X-ray crystallography[5] and MALDI mass spectrometry, pyrrolysine is made up of 4-methylpyrroline-5-carboxylate in amide linkage with the εN of lysine.[6]

Synthesis

Pyrrolysine is synthesized in vivo by joining two molecules of L-lysine. One molecule of lysine is first converted to (3R)-3-methyl-D-ornithine, which is then ligated to a second lysine. An NH2 group is eliminated, followed by cyclization and dehydration step to yield L-pyrrolysine.[7]

Catalytic function

The extra pyrroline ring is incorporated into the active site of several methyltransferases, where it is believed to rotate relatively freely. It is believed that the ring is involved in positioning and displaying the methyl group of methylamine for attack by a corrinoid cofactor. The proposed model is that a nearby carboxylic acid bearing residue, glutamate, becomes protonated, and the proton can then be transferred to the imine ring nitrogen, exposing the adjacent ring carbon to nucleophilic addition by methylamine. The positively charged nitrogen created by this interaction may then interact with the deprotonated glutamate, causing a shift in ring orientation and exposing the methyl group derived from the methylamine to the binding cleft where it can interact with corrinoid. In this way a net is transferred to the cofactor's cobalt atom with a change of oxidation state from I to III. The methylamine-derived ammonia is then released, restoring the original imine.

Genetic coding

Unlike posttranslational modifications of lysine such as hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated during translation (protein synthesis) as directed by the genetic code, just like the standard amino acids. It is encoded in mRNA by the UAG codon, which in most organisms is the 'amber' stop codon. This requires only the presence of the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon, and the pylS gene, which encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with pyrrolysine.

This novel tRNA-aaRS pair ("orthogonal pair") is independent of other synthetases and tRNAs in Escherichia coli, and further possesses some flexibility in the range of amino acids processed, making it an attractive tool to allow the placement of a possibly wide range of functional chemical groups at arbitrarily specified locations in modified proteins.[8] [9] For example, the system provided one of two fluorophores incorporated site-specifically within calmodulin to allow the real-time examination of changes within the protein by FRET spectroscopy,[10] and site-specific introduction of a photocaged lysine derivative.[11] (See Expanded genetic code)

It was originally proposed that a specific downstream sequence "PYLIS", forming a stem-loop in the mRNA, forced the incorporation of pyrrolysine instead of terminating translation in methanogenic archaea. This would be analogous to the SECIS element for selenocysteine incorporation. However, the PYLIS model has lost favor in view of the lack of structural homology between PYLIS elements and the lack of UAG stops in those species.[12]

Evolution

The pylT (tRNA) and pylS (aa-tRNA synthase) genes are part of an operon of Methanosarcina barkeri, with homologues in other sequenced members of the Methanosarcinaceae family: M. acetivorans, M. mazei, and M. thermophila. Pyrrolysine-containing proteins are known to include monomethylamine methyltransferase (mtmB), dimethylamine methyltransferase (mtbB), and trimethylamine methyltransferase (mttB). Homologs of pylS and pylT have also been found in an Antarctic archaeon, Methanosarcina barkeri and a Gram-positive bacterium, Desulfitobacterium hafniense.[13] [14] The other genes of the Pyl operon mediate pyrrolysine biosynthesis, leading to description of the operon as a "natural genetic code expansion cassette".[15]

A number of evolutionary scenarios have been proposed for the pyrrolysine system. The current (2022) view, given available sequences for tRNA and Pyl-tRNA (PylRS) synthase genes, is that:[16]

Earlier evolutionary scenarios were limited by the taxonomic range of known synthases:

Potential for an alternative translation

The tRNA(CUA) can be charged with lysine in vitro by the concerted action of the M. barkeri Class I and Class II lysyl-tRNA synthetases, which do not recognize pyrrolysine. Charging a tRNA(CUA) with lysine was originally hypothesized to be the first step in translating UAG amber codons as pyrrolysine, a mechanism analogous to that used for selenocysteine. More recent data favor direct charging of pyrrolysine on to the tRNA(CUA) by the protein product of the pylS gene, leading to the suggestion that the LysRS1:LysRS2 complex may participate in a parallel pathway designed to ensure that proteins containing the UAG codon can be fully translated using lysine as a substitute amino acid in the event of pyrrolysine deficiency.[20] Further study found that the genes encoding LysRS1 and LysRS2 are not required for normal growth on methanol and methylamines with normal methyltransferase levels, and they cannot replace pylS in a recombinant system for UAG amber stop codon suppression.[21]

Further reading

External links

Notes and References

  1. Web site: Nomenclature and Symbolism for Amino Acids and Peptides . IUPAC-IUB Joint Commission on Biochemical Nomenclature . 1983 . 5 March 2018 . https://web.archive.org/web/20081009023202/http://www.chem.qmul.ac.uk/iupac/AminoAcid/AA1n2.html . 9 October 2008 . dead .
  2. Web site: Newsletter 2009. Biochemical Nomenclature Committee of IUPAC and NC-IUBMB. Richard Cammack. 2009. Pyrrolysine. 2012-04-16. https://web.archive.org/web/20170912194130/http://www.chem.qmul.ac.uk/iubmb/newsletter/2009.html#item35. 2017-09-12. dead.
  3. Selenocysteine, Pyrrolysine, and the Unique Energy Metabolism of Methanogenic Archaea. Archaea. 2010-01-01. 1472-3646. 2933860. 20847933. 2010. 10.1155/2010/453642. Michael. Rother. Joseph A.. Krzycki. 1–14. free.
  4. Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding specialized tRNA . Science . 2002-05-24. 296. 5572. 1459–1462. 10.1126/science.1069588 . 12029131 . 2002Sci...296.1459S . Srinivasan . G . James . C. M. . Krzycki . J. A. . 28593085 .
  5. A New UAG-Encoded Residue in the Structure of a Methanogen Methyltransferase. Science. 2002-05-24. 296. 5572. 1462–1466. 10.1126/science.1069556. 12029132. 2002Sci...296.1462H . Gong. Ferguson. James. Krzycki. Chan. Hao. Bing. 35519996.
  6. The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases. 2005-11-04. 10.1074/jbc.M506402200. 280. 36962–36969. 16096277. 44. The Journal of Biological Chemistry. Soares. J. A.. Zhang. L. Pitsch. R. L.. Kleinholz. N. M.. Jones. R. B.. Wolff. J. J.. Amster. J. Green-Church. K. B.. Krzycki. J. A.. free.
  7. The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine . Nature . March 31, 2011 . 471. 7340. 647–50. 10.1038/nature09918 . 21455182 . 3070376 . 2011Natur.471..647G . Zhang . Green-Church . Krzycki . Gaston . Marsha A. .
  8. 15380192 . 10.1016/j.chembiol.2004.07.011 . 11 . 9 . Reactivity and chemical synthesis of L-pyrrolysine – the 22nd genetically encoded amino acid . September 2004 . Chemistry & Biology. 1317–24 . Hao . B . Zhao . G . Kang . P. T. . Soares . J. A. . Ferguson . T. K. . Gallucci . J . Krzycki . J. A. . Chan . M. K. . free .
  9. 19063902 . 10.1016/j.jmb.2008.11.032 . 385 . 4 . Specificity of pyrrolysyl-tRNA synthetase for pyrrolysine and pyrrolysine analogs . January 2009 . Journal of Molecular Biology. 1156–64 . Li . W. T. . Mahapatra . A . Longstaff . D. G. . Bechtel . J . Zhao . G . Kang . P. T. . Chan . M. K. . Krzycki . J. A. .
  10. 19156778 . 10.1002/anie.200805420 . 48 . 9 . A pyrrolysine analogue for protein click chemistry . 2009 . Angewandte Chemie International Edition in English. 1633–5 . Fekner . T . Li . X . Lee . M. M. . Chan . M. K. .
  11. 19378306 . 10.1002/anie.200900683 . 48 . 22 . A facile system for encoding unnatural amino acids in mammalian cells . 2873846 . 2009 . Angewandte Chemie International Edition in English. 4052–5 . Chen . P. R. . Groff . D . Guo . J . Ou . W . Cellitti . S . Geierstanger . B. H. . Schultz . P. G. .
  12. Namy. Olivier. Zhou. Yu. Gundllapalli. Sarath. Polycarpo. Carla R.. Denise. Alain. Rousset. Jean-Pierre. Söll. Dieter. Ambrogelly. Alexandre. Adding pyrrolysine to the Escherichia coli genetic code. FEBS Letters. November 2007. 581. 27. 5282–5288. 17967457. 10.1016/j.febslet.2007.10.022. free.
  13. Reviewed in Pyrrolysine and selenocysteine use dissimilar decoding strategies. Journal of Biological Chemistry. May 27, 2005. 280. 20740–20751. 10.1074/jbc.M501458200. 15788401. 21. Zhang. Y. Baranov. P. V.. Atkins. J. F.. Gladyshev. V. N.. free.
  14. High content of proteins containing 21st and 22nd amino acids, selenocysteine and pyrrolysine, in a symbiotic deltaproteobacterium of gutless worm Olavius algarvensis. Nucleic Acids Research. 2007. 35. 4952–4963. 15. 10.1093/nar/gkm514. 17626042. 1976440. Zhang. Y. Gladyshev. V. N..
  15. A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine. Proceedings of the National Academy of Sciences of the United States of America. 2007-01-16. 104. 1021–6. 3. 10.1073/pnas.0610294104. 17204561. 1783357. 2007PNAS..104.1021L . Longstaff. D. G.. Larue. R. C.. Faust. J. E.. Mahapatra. A. Zhang. L. Green-Church. K. B.. Krzycki. J. A.. free.
  16. Guo . LT . Amikura . K . Jiang . HK . Mukai . T . Fu . X . Wang . YS . O'Donoghue . P . Söll . D . Tharp . JM . Ancestral archaea expanded the genetic code with pyrrolysine. . The Journal of Biological Chemistry . November 2022 . 298 . 11 . 102521 . 10.1016/j.jbc.2022.102521 . 36152750. free . 9630628 .
  17. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proceedings of the National Academy of Sciences of the United States of America. 10.1073/pnas.0611634104. 2007-02-27. 104. 3141–3146. 9. 17360621. 1805618. 2007PNAS..104.3141A . Ambrogelly. A. Gundllapalli. S. Herring. S. Polycarpo. C. Frauer. C. Söll. D. free.
  18. Pyrrolysyl-tRNA synthetase:tRNAPyl structure reveals the molecular basis of orthogonality. Nature. 2009-02-26. 457. 1163–1167. 10.1038/nature07611. 2648862. 19118381. 7233. 2009Natur.457.1163N . Nozawa. K. O'Donoghue. P. Gundllapalli. S. Araiso. Y. Ishitani. R. Umehara. T. Söll. D. Nureki. O.
  19. Book: 19271184 . 10.1007/978-1-60327-853-9_9 . 532 . Horizontal gene transfer and the evolution of methanogenic pathways . 2009 . 163–79 . Methods in Molecular Biology . 978-1-60327-852-2 . Fournier . G . Horizontal Gene Transfer .
  20. 10.1073/pnas.0405362101. 515082. An aminoacyl-tRNA synthetase that specifically activates pyrrolysine. 15314242. Proceedings of the National Academy of Sciences of the United States of America. 2004-08-24. 101. 34. 12450–12454. 2004PNAS..10112450P . Polycarpo. C. Ambrogelly. A. Bérubé. A. Winbush. S. M.. McCloskey. J. A.. Crain. P. F.. Wood. J. L.. Söll. D. free.
  21. 17542922 . 10.1111/j.1365-2958.2007.05740.x . 64 . 5 . Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp . June 2007 . Molecular Microbiology. 1306–18 . Mahapatra . A . Srinivasan . G . Richter . K. B. . Meyer . A . Lienard . T . Zhang . J. K. . Zhao . G . Kang . P. T. . Chan . M . Gottschalk . G . Metcalf . W. W. . Krzycki . J. A. . 26445329 .

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