From Proteopedia

Revision as of 02:28, 26 March 2010

Template:STRUCTURE 2zin

Introduction

Pyrrolysyl-tRNA synthetase (PyIRS) is a group of enzymatic proteins encoded by the gene pyIS with the intention of the cellular process of tRNA aminoacylation proposed for protein translation.^[1] In particular, it purpose serves for the esterificiation of the amino acid pyrrolysine to its specific tRNA (tRNAPyl) which is post-translational modified. Pyrrolysine (Pyl) is the 22nd existing amino acid that is genetically encoded in nature and is utilized by a variety of methanogenic Archaea of the family Methanosarcinace;, along with two known bacterium species who metabolize methylamines for acquiring their energy.^[2] Pyrrolysine’s structural makeup consists of 4-methylpyrroline-5-carboxylate in amide linkage with the ϵN of lysine.^[3] This arrangement is comparable to lysine; however, being its derivative it includes a pyrroline ring situated at the back of the lysine backbone side chain. The first sighting of pyrrolysine was discovered as a byproduct contained by the active site of monomethylamine methyltransferase exclusively from Methanosarcina barkeri (M. barkeri) species.^[4] The involvement of PyIRS involvement in the ester reaction of pyrrolysine to tRNAPyl is carried out due to the anticodon CUA on the suppressor tRNAPyl that is complementary to the UAG codon.^[5][6] The interesting fact is that this is done by the response of the codon UAG (amber codon) on the mRNA that is normally a stop codon in other organisms.

In addition, by observing M. barkeri cellular mechanisms containing PyIRS it was detected that it furthermore has the capability to activate an assortment of other non-natural pyrrolysine/lysine derivatives; as well as the non-canonical amino acids.^[7] These amino acids can then be further esterified to their specialized tRNAPyl; resulting in them forming new polypeptides. This can give use proteins with manipulated structures and functions that can serve useful purposes in studying cellular processes and in altering further mechanisms. For the incorpration of these unusual amino acids PyIRS and the amber suppressor tRNAPyl are extracted from Methanosarcina and are carefully placed in to bacteria such as Escherichia coli (E. coli).^[8] This can be successful because tRNAPyl obtained from Methanosarcina function as an orthogonal pair aaRS-tRNA in E-coli without interfering with cellular mechanisms and other components of translation.^[9][10] For this to be performed for a specific amino acid a scientifically modified tRNA/aminoacyl-tRNA synthetase (aaRS) pair must be designed for the recognition and for the unique aminoacylation intended for the selected amino acid._[6] In particular, one of the many examples of non-natural amino acids include Ne-(tert-butyloxycarbonyl –L-lysine (BocLys), which is a derivative if lysine, can be intergraded into polypeptides in E.coli utilizing the amber codon by the process of being esterified to tRNAPyl by PyIRS._[4]

Structure

Pyrrolysyl-tRNA synthetase (PyIRS) proposed structure consists of a multidomain polypeptide made of 1 chain (Chain A) comprising of a total length of 291 residues. Its structure attached to BocLys, revealed above, consists of 9 α-helices (95 residues) making 32% of the structure, and the remaining is 12-β strands (59 residues) consisting of 20% of the other structure. PyIRS (c270) attached with BocLys along with and adenosine 5’ (beta, gamma-imido) triphosphate (AMMPPNP) crystal structure demonstrates the common aspects for the efficient recognition of amino acids and aminoacylation by PyIRS._[4] This complex was determined at 1.79 Anstroms and with the visible electron density in the active site it was experimentally trialed that the Ne-Boc group is situated in the hydrophobic interior similarly to the standard pyrrolysine AMPPNP bound arrangement._[4] Having the Ne-BocLys positioned in this way it has the capability to hydrogen bonds with the amide group side chain of Asn346._[4] Next, the Cα-carbonyl groups of BocLys in turn will hydrogen bond Asn346 contrary to the α-amino group which is linked to α-phosphate group of AMPPNP._[4] In order for the substrate to effectively bind to the side chain amide group of Asn354 will inducible fit the carbonyl group of the substrate pyrrolysine and BocLys into position._[4] Additionally, the BocLys α-carboxyl group is directed to that it is associated outside from the active site allowing for the flexibility due to the ability to rotate around the Cɑ- Cβ bond._[4]

Mechanism

In order for the insertion of a non-amino acids the area in the PyIRS the active sites are mutated so as to accept a non- natural amino acid, which are generally synthesized in the lab._[6] Once this has been completed aminoacylation by PyITS must take place by the activation of the chosen amino acid by the exchange of ATP for AMP and PPi (inorganic pyrophosphate). Next, the aminoacyl-AMP recognizes a specific tRNA molecule and the amino acid are transferred releasing AMP forming an aminoacyl-tRNA. In order for PyIRS to correctly recognize the appropriate amino acid there must be Ne-carbonyl group that has a specific size for the substituent._[4] To ensure the appropriate the amino acids find its way to PyIRS and not to any other class II aaRS present, PyIRS has special identification mechanisms associated it. These include specific characteristics such as its size, bulkiness according how it is going to bind to the hydrophobic position._[4] In addition, appropriate hydrogen bond acceptors/donors must be positioned adjacent Asn346, and the appropriate length of the side-chain spacer must be available to make certain the binding is excellent for the reaction to proceed efficiently._[4]

References

1. ↑ Herring S, Ambrogelly A, Polycarpo CR, Soll D. Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase. Nucleic Acids Res. 2007;35(4):1270-8. Epub 2007 Jan 31. PMID:17267409 doi:10.1093/nar/gkl1151
2. ↑ Nozawa K, O'Donoghue P, Gundllapalli S, Araiso Y, Ishitani R, Umehara T, Soll D, Nureki O. Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality. Nature. 2009 Feb 26;457(7233):1163-7. Epub 2008 Dec 31. PMID:19118381 doi:10.1038/nature07611
3. ↑ Soares JA, Zhang L, Pitsch RL, Kleinholz NM, Jones RB, Wolff JJ, Amster J, Green-Church KB, Krzycki JA. The residue mass of L-pyrrolysine in three distinct methylamine methyltransferases. J Biol Chem. 2005 Nov 4;280(44):36962-9. Epub 2005 Aug 11. PMID:16096277 doi:10.1074/jbc.M506402200
4. ↑ Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol. 2008 Nov 24;15(11):1187-97. PMID:19022179 doi:10.1016/j.chembiol.2008.10.004
5. ↑ Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol. 2008 Nov 24;15(11):1187-97. PMID:19022179 doi:10.1016/j.chembiol.2008.10.004
6. ↑ Hollman KW, O'Donnell M, Erpelding TN. Mapping elasticity in human lenses using bubble-based acoustic radiation force. Exp Eye Res. 2007 Dec;85(6):890-3. Epub 2007 Sep 22. PMID:17967452 doi:10.1016/j.exer.2007.09.006
7. ↑ Polycarpo C, Ambrogelly A, Berube A, Winbush SM, McCloskey JA, Crain PF, Wood JL, Soll D. An aminoacyl-tRNA synthetase that specifically activates pyrrolysine. Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12450-4. Epub 2004 Aug 16. PMID:15314242 doi:10.1073/pnas.0405362101
8. ↑ Namy O, Zhou Y, Gundllapalli S, Polycarpo CR, Denise A, Rousset JP, Soll D, Ambrogelly A. Adding pyrrolysine to the Escherichia coli genetic code. FEBS Lett. 2007 Nov 13;581(27):5282-8. Epub 2007 Oct 23. PMID:17967457 doi:10.1016/j.febslet.2007.10.022
9. ↑ Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem Biol. 2008 Nov 24;15(11):1187-97. PMID:19022179 doi:10.1016/j.chembiol.2008.10.004
10. ↑ Hollman KW, O'Donnell M, Erpelding TN. Mapping elasticity in human lenses using bubble-based acoustic radiation force. Exp Eye Res. 2007 Dec;85(6):890-3. Epub 2007 Sep 22. PMID:17967452 doi:10.1016/j.exer.2007.09.006