Pyrrolysyl-tRNA synthetase

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Contents

Pyrrolysyl-tRNA synthetase

Introduction

Pyrrolysyl-tRNA synthetase(PyIRS) is encoded by the gene pyIS and is founn to belong as a part of the group of enzymatic proteins whose role invovlves the cellular process of tRNA aminoacylation required for protein translation.[1] In particular, PyIRS is required for the activation of the amino acid pyrrolysine as it associates with a tRNA generating a specific tRNAPyl which is then further used to transfer the amino acid to a growing polypeptide.[2] The involvement of PyIRS is carried out due to the anticodon CUA on the supressor tRNAPyl that is complementary to the UAG codon.[3][4] 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. Pyrrolysine (Pyl) is the 22nd existing amino acid genetically encoded in nature that was first discovered as a byproduct contained by the active site of monomethylamine methyltransferase exclusively from Methanosarcina barkeri (M. barkeri) species.[2][5] Thus, it is utalized by a variety of organisms that metabolize methylamines for aquiring energy such as methanogenic Archaea of the family Methanosarcinace; along with two known bacterium species[1][5] Pyrrolysine’s structural makeup consists of 4-methylpyrroline-5-carboxylate in amide linkage with the ϵN of lysine.[6] This arrangement is comparable to lysine; however; however, being its derivative it contains an added pyrroline ring situated at the back of the structure.[6]


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 added to their specialized tRNAPyl; resulting in them forming new polypeptides. This procedure is performed my extracting PyIRS, and the amber suppressor tRNAPyl from Methanosarcina. When obtained 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.[3] Once this has been completed it then is carefully placed into a bacterium species such as Escherichia coli (E. coli).[3] The reason that this is successful is because once inserted tRNAPyl function as a orthogonal pair with aaRS-tRNA that will not interfere with cellualr mechanisms and other components of translation.[2][3] Some of the lysine derivatives such as AcLys, ZLys, BocLys, AlocLys and AzZLys have been experimentally trialed and as a result, have been successfully translated into proteins.[3] In particular interest, Nϵ-(tert-butyloxycarbonyl –L-lysine (BocLys)is a non-natural amino acid that is a deviation from the structure of lysine which can be intergraded into polypeptides utilizing the amber codon by the process of being esterified to tRNAPyl by PyIRS in E.coli for the incorporation into proteins.[2] By using this method we can obtain proteins with manipulated structures and functions that can serve useful purposes in studying cellular processes and in altering further mechanisms.

Structure

Figure 1: Showing the chains

Drag the structure with the mouse to rotate
Pyrrolysyl-tRNA synthetase (PyIRS) attached with , and along with adenosine 5’ (beta, gamma-imido) triphosphate (AMMPPNP)demonstrates now known structural components and configuration that is needed for the efficient recognition of amino acids and the aminoacylation by PyIRS.[2] As shown in figure 1, it consists of a multidomain polypeptide made of 1 chain (Chain A) comprising of a total length of 291 residues. The 2 catalytic domains are PRK06253 and class_II_aaRS-like_core that are used for the fermentation of the enzyme bound aminoacyl-adenylate in the presence of ATP. Furthermore, The structure is observed to consisting of 9 (95 residues) making 32% of the structure, and the remaining is 12 (59 residues) consisting of 20% of the other structure. In addition, its structure components require the presence of the 4 ligands; ANP, EDO, LBY, and MG for the protein to correctly perform its biological function. This complex was determined at 1.79 Anstroms using the Fo-Fc omit map and with the visible electon density in the active site it was experimentally trialed that the Nϵ-Boc group is situated in the hydrophobic interior, in the similar was as the already observed traditional pyrrolysine AMPPNP bound arrangement.[2] For that reason having the Nϵ-BocLys positioned in this way has the capability to participate in the hydrogen bonding with Asn346 amide group creating a suitable environment for the binding.[2] 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.[2] In order for the substrate to effectively bind to the side chain amide group Asn354 will inducible fit the carbonyl group of the substrate pyrrolysine and BocLys into position.[2] 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.[2]

Mechanism

Th proposed mechasim for the insertion of pyrrolysine into polypeptides initially begins with the activation of a special tRNA.[8]. This is completed by the charging of tRNA as it interacts with lysine via aminoacylation by PyIRS in the presence of the exchange of ATP for AMP and PPi (inorganic pyrophosphate).[8] This will generate lysyl-tRNACUA which is then pre-translationally customized by the influence of the genes PyIB, PyIC and PyID generating Pyl-tRNACU.[8] This specific tRNAPyl is used to transport the amino acid pyrrolysine to the A-site located in the ribosome which is then added to the co- translated polypeptide chain.[8] To ensure the appropriate amino acid find its way to PyIRS, and not to any other class II aaRS present, PyIRS has special identification systems associated with it.[2] These include special features such as its overall dimension and structural layout and its binding ability to the hydrophibic active site [2] For the accurate recognition of the appropriate amino acid there must be Ne-carbonyl group that has a specific size for the substituent.[2] In addition, as it is being bound there must be appropriate hydrogens able to donate or accept electrons positioned adjacent to Asn346, and further as this binding proceeds there must be side chain spacers with the appropriate length to make certain the binding is excellent for the reaction to proceed efficiently.[2] For the proper recognition of the appropriate amino acid, it must contains a Nϵ-carbonyl group that has a specific size for the substituent.[2]

References

  1. ↑ 1.0 1.1 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. ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 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
  3. ↑ 3.0 3.1 3.2 3.3 3.4 Urbancsek J, Rabe T, Grunwald K, Kiesel L, Papp Z, Runnebaum B. High preovulatory serum luteinizing hormone level is unfavorable to conception. Gynecol Endocrinol. 1991 Dec;5(4):223-33. PMID:1796745
  4. ↑ 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
  5. ↑ 5.0 5.1 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
  6. ↑ 6.0 6.1 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
  7. ↑ Polycarpo CR, Herring S, Berube A, Wood JL, Soll D, Ambrogelly A. Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase. FEBS Lett. 2006 Dec 11;580(28-29):6695-700. Epub 2006 Nov 20. PMID:17126325 doi:10.1016/j.febslet.2006.11.028
  8. ↑ 8.0 8.1 8.2 8.3 Ibba M, Soll D. Genetic code: introducing pyrrolysine. Curr Biol. 2002 Jul 9;12(13):R464-6. PMID:12121639
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