Function
Pyrrolysyl-tRNA synthetase (PyIRS) is encoded by the gene pyIS and is found to belong as a part of the group of enzymatic proteins whose role involves 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 suppressor 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 utilized by a variety of organisms that metabolize methylamines for acquiring 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 Nϵ of lysine.[6] This arrangement is comparable to lysine; however, being its derivative it contains an added pyrroline ring that is found to lie 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 imitative of pyrrolysine/lysine; as well as amino acids that are non-canonical.[7] These amino acids can then be further added to their specialized tRNAPyl; inventing new polypeptides.[5] This procedure is performed by extracting PyIRS, and the amber suppressor tRNAPyl from Methanosarcina. When obtained it must be ensured that the PyIRS-tRNAPyl pair are going to perform their specific roles with the selected amino acids to produce polypeptides containing them.[3] Thus, their structural components are manipulated and then experimentally designed for the recognition and for the unique aminoacylation intended for it.[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 an orthogonal pair with aaRS-tRNA that will not interfere with cellular 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. See also Ligases.
Structure
(2zin).
Pyrrolysyl-tRNA synthetase (PyIRS) catalytic complex attached with , and along with adenosine 5’ (beta, gamma-imido) triphosphate (AMMPPNP) demonstrates the 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 multi domain polypeptide made of 1 chain (Chain A) comprising of a total length of 291 residues with 2 catalytic domains; PRK06253 and class_II_aaRS-like_core t.[2] 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.
PyIRS structure is found to enclose a hydrophobic interior where the catalytic activity of recognition and activation of the amino acids is going to take place.[8] This area creates a suitable environment for the binding to occur and for accommodating the residues of pyrrolysine methyl-pyrroline ring inside so it has the capability so further interact with the active-side residues available.[8] It has been shown, that in order for the amino acid to successfully come in contact proper size of PyIRS must be incorporated at the Nϵ-carbonyl group.[2] By using the Fo-Fc omit map and with the visible electron 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 it has the capability to participate in the hydrogen bonding with Asn346 amide group.[2] This Asn346 is important when the amino acid is binding because it helps attach its carbonyl side chain; as well as the main-chain α-amino group to ensure the proper recognition it going to take place.[8] From this Asn346 will also allow the substrate to effectively bind to the side chain amide group by inducible fitting the carbonyl group of the substrate into position.[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] 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] Furthermore, this active site contains an abundant of functional residues such as Lys192, Arg197, Arg217, Lys336, Lys435, Lys438 and Arg439 from the one domain and Arg310, Lys311 and Arg314 from the other, which participate in the effective binding with the tRNA.[8]
Mechanism
The proposed mechanism for the insertion of pyrrolysine into polypeptides initially begins with the activation of a special tRNA.[9] This is completed by the charging of the 3' CCA end 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).[9] [8] When ATP binds to PyIRS it causes a conformational change allowing this to happen.[8] This will generate lysyl-tRNACUA which is then pre-translationally customized by the influence of the genes PyIB, PyIC and PyID generating Pyl-tRNACUA[9] 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.[9] 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, its structural layout and its binding ability to the hydrophibic active site.[2]
