|Gene:||ileS||Accession Numbers: EG10492 (EcoCyc), b0026, ECK0027|
Synonyms: ilvS, IleRS
Isoleucyl-tRNA synthetase (IleRS) is a member of the family of aminoacyl-tRNA synthetases, which interpret the genetic code by covalently linking amino acids to their specific tRNA molecules. The reaction is driven by ATP hydrolysis. IleRS belongs to the Class I aminoacyl tRNA synthetases [Eriani90, Landes95].
Specificity determinants within tRNAIle that are important for recognition by IleRS have been identified [Schoemaker76, Schoemaker77, Schoemaker77a, Nureki91, Pallanck91, Niimi93, Nureki94, Salowe09]. Residues within IleRS that interact with tRNAIle have been identified [Schmidt95, Auld96]. IleRS specifically acylates the 2'-OH and deacylates the 3'-OH of tRNAIle [Nordin02]. The aminoacyl linkage between Ile and tRNAIle is the most stable among the 12 pairs of cognate amino acid-tRNA pairs measured by [Peacock14].
IleRS contains two zinc atoms per active site [Xu94a]. One zinc atom bound near the N-terminal catalytic core is important for amino acid binding and utilization [Landro94]. The second zinc binding site has been mapped near the C terminus of IleRS [Landro94a] and is required for the tRNA binding step in aminoacylation [Zhou95a]. The C-terminal peptide containing this site can complement the specific tRNA binding and catalytic activity of the N-terminal domain in trans [Glasfeld96, Glasfeld97]. If separately expressed, the three domains of IleRS can form an active complex and complement each other in trans [Shiba92, Shiba92a]. The dispensable CP1 domain interrupts the nucleotide binding domain within IleRS [Starzyk87]. IleRS binds one molecule of tRNA per molecule of enzyme [Hustedt77]. Affinity labeling experiments were used to identify various binding and active sites of IleRS [Baouz09].
Binding specificity of IleRS for various aliphatic amino acids and aminoalkyl adenylates has been determined; none of the tested isoleucine analogs bind as tightly as isoleucine itself [Flossdorf76, Hinz76, Flossdorf77, Kohda87]. Both amino acid discrimination and proofreading contribute to the specificity of Ile charging onto tRNAIle [Freist87, Freist88], and experimental data is consistent with the existence of both a pre- and a post-transfer editing capability of the enzyme, depending on the amino acid [Fersht77, Yamane77, Freist87a, Cramer91]. Misactivated homocysteine undergoes pretransfer editing [Jakubowski81]. Although valine is discriminated against at the amino acid recognition stage, IleRS misactivates valine at a significant rate. A single point mutation, G56A, eliminates the ability to discriminate against valine, and valine is then discriminated against by hydrolytic editing [Schmidt94]. The presence of misacylated tRNA is thought to be required for both pre- and post-transfer editing [Nordin03].
IleRS appears to be the only class I enzyme for which pre-transfer editing is important. The rates of hydrolysis of a pre-transfer amino-adenylate and a misacylated tRNA are approximately equal for IleRS [Dulic10]. The tRNA-dependent pre-transfer editing activity accounts for approximately 1/3 of the total proofreading activity. The conserved Tyr59 residue within the tRNA aminoacylation active site is involved in tRNA-dependent pre-transfer editing [Dulic14]. The editing response appears to be triggered by specific bases in the effector and does not require a tRNA-like structure or active acceptor hydroxyl groups [Hale96]. Pre-transfer editing was initially thought to involve translocation of the misactivated amino acid from the active site to the editing site [Nomanbhoy99, Farrow01, Bishop02a, Hendrickson02, Bishop03].
The post-transfer editing site maps to the CP1 domain, an insertion that interrupts the Rossman fold nucleotide-binding domain [Schmidt95]. The isolated CP1 domain alone has post-transfer editing activity [Lin96]. The editing site is distinct from the active site for aminoacylation [Hendrickson00]. Distinct residues within the editing site are required for editing the misactivated adenylate form and the aminoacyl ester [Hendrickson02].
Site-directed mutagenesis identified the isoleucine binding site [Clarke88]. Labeling studies with pyridoxal-5'-phosphate identified residues that may be important for binding of the phosphates of ATP [Kalogerakos94]. An editing-deficient IleRS mutant strain has a growth yield advantage under specialized conditions, generating "statistical proteins" containing norvaline [Pezo04], but under most conditions, it has a lower growth rate [Bacher05].
|Map Position: [22,391 -> 25,207] (0.48 centisomes, 2°)||Length: 2817 bp / 938 aa|
Molecular Weight of Polypeptide: 104.3 kD (from nucleotide sequence), 102 kD (experimental) [Durekovic73 ]
Unification Links: ASAP:ABE-0000094 , CGSC:613 , DIP:DIP-10017N , EchoBASE:EB0487 , EcoGene:EG10492 , EcoliWiki:b0026 , Mint:MINT-1232480 , ModBase:P00956 , OU-Microarray:b0026 , PortEco:ileS , PR:PRO_000023000 , Pride:P00956 , Protein Model Portal:P00956 , RefSeq:NP_414567 , RegulonDB:EG10492 , SMR:P00956 , String:511145.b0026 , UniProt:P00956
Relationship Links: InterPro:IN-FAMILY:IPR001412 , InterPro:IN-FAMILY:IPR002300 , InterPro:IN-FAMILY:IPR002301 , InterPro:IN-FAMILY:IPR009008 , InterPro:IN-FAMILY:IPR009080 , InterPro:IN-FAMILY:IPR010663 , InterPro:IN-FAMILY:IPR013155 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR023585 , Panther:IN-FAMILY:PTHR11946:SF9 , Pfam:IN-FAMILY:PF00133 , Pfam:IN-FAMILY:PF06827 , Pfam:IN-FAMILY:PF08264 , Prints:IN-FAMILY:PR00984 , Prosite:IN-FAMILY:PS00178
In Paralogous Gene Group: 7 (4 members)
|Biological Process:||GO:0006428 - isoleucyl-tRNA aminoacylation
[GOA06, GOA01a, Fersht76]
GO:0006412 - translation [UniProtGOA11]
GO:0006418 - tRNA aminoacylation for protein translation [GOA01a]
GO:0006450 - regulation of translational fidelity [GOA01a]
GO:0046677 - response to antibiotic [UniProtGOA11]
|Molecular Function:||GO:0004822 - isoleucine-tRNA ligase activity
[GOA06, GOA01, GOA01a, Durekovic73, Fersht76]
GO:0008270 - zinc ion binding [GOA06, Xu94a]
GO:0000166 - nucleotide binding [UniProtGOA11, GOA01a]
GO:0002161 - aminoacyl-tRNA editing activity [GOA01a]
GO:0003824 - catalytic activity [GOA01a]
GO:0004812 - aminoacyl-tRNA ligase activity [UniProtGOA11, GOA01a]
GO:0005524 - ATP binding [UniProtGOA11, GOA06, GOA01a]
GO:0016874 - ligase activity [UniProtGOA11]
GO:0046872 - metal ion binding [UniProtGOA11]
|Cellular Component:||GO:0005829 - cytosol
[Gaudet10, DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm [UniProtGOA11a, UniProtGOA11, GOA06, GOA01a]
|MultiFun Terms:||information transfer → protein related → amino acid -activation|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB Lennox||No||37||Aerobic||7||No [Baba06, Yamamoto09]|
Enzymatic reaction of: isoleucyl-tRNA synthetase
Synonyms: isoleucine--tRNA ligase
EC Number: 126.96.36.199
The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.
The reaction is physiologically favored in the direction shown.
In Pathways: tRNA charging
Many biochemical experiments on IleRS were done with the enzyme from E. coli MRE600 (e.g. [Durekovic73]), which is not a K-12 strain, or with the E. coli B enzyme (e.g. [Baldwin66a, Schoemaker76]).
Pseudomonic acid (mupirocin) is a competitive inhibitor of IleRS from E. coli B [Hughes80].
The magnesium dependency of the equilibrium constant of IleRS has been measured [Airas07].
|Chain||2 -> 938|
|Protein-Segment||58 -> 68|
|Sequence-Conflict||243 -> 264|
|Sequence-Conflict||300 -> 301|
|Protein-Segment||602 -> 606|
|Sequence-Conflict||740 -> 743|
|Sequence-Conflict||867 -> 869|
10/20/97 Gene b0026 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10492; confirmed by SwissProt match.
Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554
Bacher05: Bacher JM, de Crecy-Lagard V, Schimmel PR (2005). "Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase." Proc Natl Acad Sci U S A 102(5);1697-701. PMID: 15647356
Baouz09: Baouz S, Schmitter JM, Chenoune L, Beauvallet C, Blanquet S, Woisard A, Hountondji C (2009). "Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry." Open Biochem J 3;26-38. PMID: 19557155
Bishop02a: Bishop AC, Nomanbhoy TK, Schimmel P (2002). "Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase." Proc Natl Acad Sci U S A 99(2);585-90. PMID: 11782529
Bishop03: Bishop AC, Beebe K, Schimmel PR (2003). "Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase." Proc Natl Acad Sci U S A 100(2);490-4. PMID: 12515858
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Dulic10: Dulic M, Cvetesic N, Perona JJ, Gruic-Sovulj I (2010). "Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases." J Biol Chem 285(31);23799-809. PMID: 20498377
Dulic14: Dulic M, Perona JJ, Gruic-Sovulj I (2014). "Determinants for tRNA-Dependent Pretransfer Editing in the Synthetic Site of Isoleucyl-tRNA Synthetase." Biochemistry 53(39);6189-98. PMID: 25207837
Eriani90: Eriani G, Delarue M, Poch O, Gangloff J, Moras D (1990). "Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs." Nature 347(6289);203-6. PMID: 2203971
Fersht76: Fersht AR, Kaethner MM (1976). "Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12." Biochemistry 15(4);818-23. PMID: 764868
Flossdorf76: Flossdorf J, Pratorius HJ, Kula MR (1976). "Influence of side-chain structure of aliphatic amino acids on binding to isoleucyl-tRNA synthetase from Escherichia coli MRE 600." Eur J Biochem 66(1);147-55. PMID: 782880
Flossdorf77: Flossdorf J, Marutzky R, Messer K, Kula MR (1977). "On the binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600." Nucleic Acids Res 4(3);673-83. PMID: 325520
Freist82: Freist W, Sternbach H, Cramer F (1982). "Isoleucyl-tRNA synthetase from Escherichia coli MRE 600. Different pathways of the aminoacylation reaction depending on presence of pyrophosphatase, order of substrate addition in the pyrophosphate exchange, and substrate specificity with regard to ATP analogs." Eur J Biochem 128(2-3);315-29. PMID: 6129973
Freist87: Freist W, Sternbach H, Cramer F (1987). "Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A(3'NH2)." Eur J Biochem 169(1);33-9. PMID: 3315663
Freist87a: Freist W, Cramer F (1987). "Isoleucyl-tRNA synthetase from Escherichia coli MRE 600: discrimination between isoleucine and valine with modulated accuracy." Biol Chem Hoppe Seyler 368(3);229-37. PMID: 3297096
Freist88: Freist W, Sternbach H, Cramer F (1988). "Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A." Eur J Biochem 173(1);27-34. PMID: 3281834
Hendrickson02: Hendrickson TL, Nomanbhoy TK, de Crecy-Lagard V, Fukai S, Nureki O, Yokoyama S, Schimmel P (2002). "Mutational separation of two pathways for editing by a class I tRNA synthetase." Mol Cell 9(2);353-62. PMID: 11864608
Hinz76: Hinz HJ, Weber K, Flossdorf J, Kula MR (1976). "Thermodynamic studies on the specificity of L-isoleucine-tRNA ligase of Escherichia coli MRE 600. Calorimetric investigations on binding of amino acids and isoleucinol to the enzyme." Eur J Biochem 71(2);437-42. PMID: 795668
Innis84: Innis MA, Tokunaga M, Williams ME, Loranger JM, Chang SY, Chang S, Wu HC (1984). "Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene." Proc Natl Acad Sci U S A 81(12);3708-12. PMID: 6374664
Kalogerakos94: Kalogerakos T, Hountondji C, Berne PF, Dukta S, Blanquet S (1994). "Modification of aminoacyl-tRNA synthetases with pyridoxal-5'-phosphate. Identification of the labeled amino acid residues." Biochimie 76(1);33-44. PMID: 8031903
Kamio85: Kamio Y, Lin CK, Regue M, Wu HC (1985). "Characterization of the ileS-lsp operon in Escherichia coli. Identification of an open reading frame upstream of the ileS gene and potential promoter(s) for the ileS-lsp operon." J Biol Chem 260(9);5616-20. PMID: 2985604
Kim77: Kim JJ, Chakraburtty K, Mehler AH (1977). "Evidence for single mechanism for aminoacyl-tRNA synthetases including aminoacyl adenylates as intermediates." J Biol Chem 252(8);2698-701. PMID: 323252
Kohda87: Kohda D, Kawai G, Yokoyama S, Kawakami M, Mizushima S, Miyazawa T (1987). "NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase." Biochemistry 26(20);6531-8. PMID: 3322383
Landes95: Landes C, Perona JJ, Brunie S, Rould MA, Zelwer C, Steitz TA, Risler JL (1995). "A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases." Biochimie 77(3);194-203. PMID: 7647112
Landro94: Landro JA, Schmidt E, Schimmel P, Tierney DL, Penner-Hahn JE (1994). "Thiol ligation of two zinc atoms to a class I tRNA synthetase: evidence for unshared thiols and role in amino acid binding and utilization." Biochemistry 33(47);14213-20. PMID: 7947832
Lee03a: Lee J, Kim SE, Lee JY, Kim SY, Kang SU, Seo SH, Chun MW, Kang T, Choi SY, Kim HO (2003). "N-Alkoxysulfamide, N-hydroxysulfamide, and sulfamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases." Bioorg Med Chem Lett 13(6);1087-92. PMID: 12643918
Miller87: Miller KW, Wu HC (1987). "Cotranscription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. Fine-structure mapping of the lsp internal promoter." J Biol Chem 262(1);389-93. PMID: 2432063
Miller87a: Miller KW, Bouvier J, Stragier P, Wu HC (1987). "Identification of the genes in the Escherichia coli ileS-lsp operon. Analysis of multiple polycistronic mRNAs made in vivo." J Biol Chem 262(15);7391-7. PMID: 3294831
Niimi93: Niimi T, Kawai G, Takayanagi M, Noguchi T, Hayashi N, Kohno T, Muto Y, Watanabe K, Miyazawa T, Yokoyama S (1993). "A 15N-1H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli." Biochimie 75(12);1109-15. PMID: 8199246
Nomanbhoy99: Nomanbhoy TK, Hendrickson TL, Schimmel P (1999). "Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis." Mol Cell 4(4);519-28. PMID: 10549284
Nordin03: Nordin BE, Schimmel P (2003). "Transiently misacylated tRNA is a primer for editing of misactivated adenylates by class I aminoacyl-tRNA synthetases." Biochemistry 42(44);12989-97. PMID: 14596614
Nureki91: Nureki O, Tateno M, Niimi T, Kohno T, Muramatsu T, Kanno H, Muto Y, Giege R, Yokoyama S (1991). "Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases." Nucleic Acids Symp Ser (25);165-6. PMID: 1726806
Nureki94: Nureki O, Niimi T, Muramatsu T, Kanno H, Kohno T, Florentz C, Giege R, Yokoyama S (1994). "Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli." J Mol Biol 236(3);710-24. PMID: 8114089
Nureki98: Nureki O, Vassylyev DG, Tateno M, Shimada A, Nakama T, Fukai S, Konno M, Hendrickson TL, Schimmel P, Yokoyama S (1998). "Enzyme structure with two catalytic sites for double-sieve selection of substrate." Science 280(5363);578-82. PMID: 9554847
Pallanck91: Pallanck L, Schulman LH (1991). "Anticodon-dependent aminoacylation of a noncognate tRNA with isoleucine, valine, and phenylalanine in vivo." Proc Natl Acad Sci U S A 88(9);3872-6. PMID: 2023934
Pezo04: Pezo V, Metzgar D, Hendrickson TL, Waas WF, Hazebrouck S, Doring V, Marliere P, Schimmel P, De Crecy-Lagard V (2004). "Artificially ambiguous genetic code confers growth yield advantage." Proc Natl Acad Sci U S A 101(23);8593-7. PMID: 15163798
Salowe09: Salowe SP, Wiltsie J, Hawkins JC, Sonatore LM (2009). "The catalytic flexibility of tRNAIle-lysidine synthetase can generate alternative tRNA substrates for isoleucyl-tRNA synthetase." J Biol Chem 284(15):9656-62. PMID: 19233850
Schimmel93: Schimmel P, Landro JA, Schmidt E (1993). "Evidence for distinct locations for metal binding sites in two closely related class I tRNA synthetases." J Biomol Struct Dyn 11(3);571-81. PMID: 8129874
Schmidt95: Schmidt E, Schimmel P (1995). "Residues in a class I tRNA synthetase which determine selectivity of amino acid recognition in the context of tRNA." Biochemistry 34(35);11204-10. PMID: 7669778
Schoemaker77: Schoemaker HJ, Schimmel PR (1977). "Inhibition of an aminoacyl transfer RNA synthetase by a specific trinucleotide derived from the sequence of its cognate transfer RNA." Biochemistry 16(25);5461-4. PMID: 336087
Shepard92: Shepard A, Shiba K, Schimmel P (1992). "RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis." Proc Natl Acad Sci U S A 89(20);9964-8. PMID: 1329109
Tokunaga85: Tokunaga M, Loranger JM, Chang SY, Regue M, Chang S, Wu HC (1985). "Identification of prolipoprotein signal peptidase and genomic organization of the lsp gene in Escherichia coli." J Biol Chem 260(9);5610-5. PMID: 2580835
Webster84: Webster T, Tsai H, Kula M, Mackie GA, Schimmel P (1984). "Specific sequence homology and three-dimensional structure of an aminoacyl transfer RNA synthetase." Science 226(4680);1315-7. PMID: 6390679
Yamamoto09: Yamamoto N, Nakahigashi K, Nakamichi T, Yoshino M, Takai Y, Touda Y, Furubayashi A, Kinjyo S, Dose H, Hasegawa M, Datsenko KA, Nakayashiki T, Tomita M, Wanner BL, Mori H (2009). "Update on the Keio collection of Escherichia coli single-gene deletion mutants." Mol Syst Biol 5;335. PMID: 20029369
Yanagisawa94: Yanagisawa T, Lee JT, Wu HC, Kawakami M (1994). "Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase." J Biol Chem 269(39);24304-9. PMID: 7929087
Zhang09: Zhang J, Sprung R, Pei J, Tan X, Kim S, Zhu H, Liu CF, Grishin NV, Zhao Y (2009). "Lysine acetylation is a highly abundant and evolutionarily conserved modification in Escherichia coli." Mol Cell Proteomics 8(2);215-25. PMID: 18723842
Zhou95a: Zhou L, Rosevear PR (1995). "Mutation of the carboxy terminal zinc finger of E. coli isoleucyl-tRNA synthetase alters zinc binding and aminoacylation activity." Biochem Biophys Res Commun 216(2);648-54. PMID: 7488160
Nonaka06: Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA (2006). "Regulon and promoter analysis of the E. coli heat-shock factor, sigma32, reveals a multifaceted cellular response to heat stress." Genes Dev 20(13);1776-89. PMID: 16818608
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