|Gene:||leuS||Accession Numbers: EG10532 (EcoCyc), b0642, ECK0635|
Leucyl-tRNA synthetase (LeuRS) 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. LeuRS belongs to the Class IB aminoacyl tRNA synthetases; apart from sequence motifs within the active site, the different enzymes show little similarity in their primary amino acid sequences [Landes95].
Correct aminoacylation by LeuRS requires both an activation and an editing function [Ibba00]. The CCA acceptor end of tRNALeu is essential for both the aminoacylation and editing reaction [Zhou11]. Measurement of kinetic parameters for both reactions showed that the rate-limiting step for posttransfer editing is product release, and that its active site functions by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA [Cvetesic12].
LeuRS shows high initial substrate discrimination and appears to correct mistakes by posttransfer editing alone [Englisch86]. LeuRS editing defects are lethal to the cell [Karkhanis06]. The CP1 (connective polypeptide 1) domain, which splits the nucleotide binding fold [Starzyk87], is required for the posttransfer editing function [Chen00a, Mursinna01, Du02]. The Thr251 residue is a critical specificity determinant [Mursinna04, Xu04c], and two conserved Thr residues within CP1 play a central role in amino acid editing, possibly by stabilizing the transition state [Zhai05]. The conserved Arg249 residue confers specific amino acid substrate recognition [Zhai07]. The β-strands as well as the short flexible "hinges" connecting the CP1 domain to the aminoacylation core are required for editing of mischarged tRNALeu [Betha07]. The effect of point mutations in the β-strands on enzyme activity support the hypothesis that the flexibility and orientation of the β-strands is essential for function [Mascarenhas08]. Mutations in both hinge regions allow mischarged tRNAs to bypass the posttransfer editing site [Hellmann09, Mascarenhas09].
The mechanistic details of posttransfer editing have been elucidated by a series of co-crystal structures of LeuRS with its substrate in the editing and aminoacylation conformations. Translocation of the 3' end of tRNAs from the aminoacylation to the editing site involves correlated rotations of four flexibly linked LeuRS domains [Palencia12]. Crystal structures of the editing domain in complex with non-cognate amino acids showed that amino acid discrimination is based on a lock-and-key mechanism [Liu06].
A pretransfer editing activity can be unmasked by secondary mutations within the CP1 domain [Williams06] or by specifically inhibiting the posttransfer editing function [Tan10]. Deletion of the entire CP1 domain results in loss of posttransfer editing and unmasks a tRNA-dependent editing activity that resides in the aminoacylation active site [Boniecki08].
Deletion of the CP2 domain (residues 194-225) leads to loss of leucine activation, aminoacylation and posttransfer editing activities of LeuRS [Zhou08a].
In contrast to results with the yeast enzyme, deletion of the C-terminal domain of E. coli LeuRS nearly abolishes aminoacylation and editing activity [Hsu06]. The length of the flexible peptide linker which connects the C-terminal domain to the main body of the enzyme is critical especially for the aminoacylation activitiy of the enzyme [Hsu08]. The Leu570 residue is important for both amino acid discrimination and tRNA binding [Lue07]. The "leucine-specific domain" (amino acids 572-617) immediately upstream of the catalytic site is required for aminoacylation activity, but not post-transfer editing [Vu07].
leuS is an essential gene [Baba06]. Strains containing an editing-defective mutant allele show a growth defect when grown on medium containing isoleucine, methionine, valine, or various non-standard amino acids [Karkhanis07].
|Map Position: [671,424 <- 674,006] (14.47 centisomes, 52°)||Length: 2583 bp / 860 aa|
Molecular Weight of Polypeptide: 97.234 kD (from nucleotide sequence)
Unification Links: ASAP:ABE-0002196 , CGSC:566 , DIP:DIP-10095N , EchoBASE:EB0527 , EcoGene:EG10532 , EcoliWiki:b0642 , Mint:MINT-1226410 , ModBase:P07813 , OU-Microarray:b0642 , PortEco:leuS , PR:PRO_000023086 , Pride:P07813 , Protein Model Portal:P07813 , RefSeq:NP_415175 , RegulonDB:EG10532 , SMR:P07813 , String:511145.b0642 , UniProt:P07813
Relationship Links: InterPro:IN-FAMILY:IPR001412 , InterPro:IN-FAMILY:IPR002300 , InterPro:IN-FAMILY:IPR002302 , InterPro:IN-FAMILY:IPR009008 , InterPro:IN-FAMILY:IPR009080 , InterPro:IN-FAMILY:IPR013155 , InterPro:IN-FAMILY:IPR014729 , InterPro:IN-FAMILY:IPR025709 , Panther:IN-FAMILY:PTHR11946:SF7 , PDB:Structure:2AJG , PDB:Structure:2AJH , PDB:Structure:2AJI , PDB:Structure:3ZGZ , PDB:Structure:3ZJT , PDB:Structure:3ZJU , PDB:Structure:3ZJV , PDB:Structure:4AQ7 , PDB:Structure:4ARC , PDB:Structure:4ARI , PDB:Structure:4AS1 , PDB:Structure:4CQN , Pfam:IN-FAMILY:PF00133 , Pfam:IN-FAMILY:PF08264 , Pfam:IN-FAMILY:PF13603 , Prints:IN-FAMILY:PR00985 , Prosite:IN-FAMILY:PS00178
In Paralogous Gene Group: 7 (4 members)
|Biological Process:||GO:0006429 - leucyl-tRNA aminoacylation
[GOA06, GOA01a, Chen99a]
GO:0006412 - translation [UniProtGOA11a]
GO:0006418 - tRNA aminoacylation for protein translation [GOA01a]
GO:0006450 - regulation of translational fidelity [GOA01a]
|Molecular Function:||GO:0004823 - leucine-tRNA ligase activity
[GOA06, GOA01, GOA01a, Chen99a]
GO:0000166 - nucleotide binding [UniProtGOA11a, GOA01a]
GO:0002161 - aminoacyl-tRNA editing activity [GOA01a]
GO:0004812 - aminoacyl-tRNA ligase activity [UniProtGOA11a, GOA01a]
GO:0005524 - ATP binding [UniProtGOA11a, GOA06, GOA01a]
GO:0016874 - ligase activity [UniProtGOA11a]
|Cellular Component:||GO:0005829 - cytosol
[Gaudet10, DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm [UniProtGOA11, UniProtGOA11a, GOA06]
|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, Comment 1]|
Enzymatic reaction of: leucyl-tRNA synthetase
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
|Protein-Segment||42 -> 52|
|Protein-Segment||619 -> 623|
10/20/97 Gene b0642 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10532; 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
Betha07: Betha AK, Williams AM, Martinis SA (2007). "Isolated CP1 domain of Escherichia coli leucyl-tRNA synthetase is dependent on flanking hinge motifs for amino acid editing activity." Biochemistry 46(21);6258-67. PMID: 17474713
Boniecki08: Boniecki MT, Vu MT, Betha AK, Martinis SA (2008). "CP1-dependent partitioning of pretransfer and posttransfer editing in leucyl-tRNA synthetase." Proc Natl Acad Sci U S A 105(49);19223-8. PMID: 19020078
Brustad08: Brustad E, Bushey ML, Brock A, Chittuluru J, Schultz PG (2008). "A promiscuous aminoacyl-tRNA synthetase that incorporates cysteine, methionine, and alanine homologs into proteins." Bioorg Med Chem Lett 18(22);6004-6. PMID: 18845434
Chen01a: Chen JF, Li T, Wang ED, Wang YL (2001). "Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase." Biochemistry 40(5);1144-9. PMID: 11170439
Chung97: Chung E., Allen E., Araujo R., Aparicio A.M., Davis K., Duncan M., Federspiel N., Hyman R., Kalman S., Komp C., Kurdi O., Lew H., Lin D., Namath A., Oefner P., Roberts D., Schramm S., Davis R.W. (1997). "Sequence of minutes 4-25 of Escherichia coli." Data submission to EMBL/GenBank/DDBJ databases on 1997-01.
Cvetesic12: Cvetesic N, Perona JJ, Gruic-Sovulj I (2012). "Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps." J Biol Chem 287(30);25381-94. PMID: 22648413
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
Englisch86: Englisch S, Englisch U, von der Haar F, Cramer F (1986). "The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast." Nucleic Acids Res 14(19);7529-39. PMID: 3534789
Hsu06: Hsu JL, Rho SB, Vannella KM, Martinis SA (2006). "Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles." J Biol Chem 281(32);23075-82. PMID: 16774921
Karkhanis06: Karkhanis VA, Boniecki MT, Poruri K, Martinis SA (2006). "A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria." J Biol Chem 281(44);33217-25. PMID: 16956879
Karkhanis07: Karkhanis VA, Mascarenhas AP, Martinis SA (2007). "Amino acid toxicities of Escherichia coli that are prevented by leucyl-tRNA synthetase amino acid editing." J Bacteriol 189(23);8765-8. PMID: 17890314
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
Liu06: Liu Y, Liao J, Zhu B, Wang E, Ding J (2006). "Crystal structures of the editing domain of E. coli leucyl-tRNA synthetase and its complexes with methionine and isoleucine reveal a lock-and-key mechanism for amino acid discrimination." Biochem J 394(Pt 2):399-407. PMID: 16277600
Mascarenhas08: Mascarenhas AP, Martinis SA (2008). "Functional segregation of a predicted "hinge" site within the beta-strand linkers of Escherichia coli leucyl-tRNA synthetase." Biochemistry 47(16):4808-16. PMID: 18363380
Mascarenhas09: Mascarenhas AP, Martinis SA (2009). "A glycine hinge for tRNA-dependent translocation of editing substrates to prevent errors by leucyl-tRNA synthetase." FEBS Lett 583(21);3443-7. PMID: 19796639
Mursinna01: Mursinna RS, Lincecum TL, Martinis SA (2001). "A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu." Biochemistry 40(18);5376-81. PMID: 11331000
Mursinna04: Mursinna RS, Lee KW, Briggs JM, Martinis SA (2004). "Molecular dissection of a critical specificity determinant within the amino acid editing domain of leucyl-tRNA synthetase." Biochemistry 43(1);155-65. PMID: 14705941
Palencia12: Palencia A, Crepin T, Vu MT, Lincecum TL, Martinis SA, Cusack S (2012). "Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase." Nat Struct Mol Biol 19(7);677-84. PMID: 22683997
Tan12: Tan M, Yan W, Liu RJ, Wang M, Chen X, Zhou XL, Wang ED (2012). "A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity." Biochem J 443(2);477-84. PMID: 22292813
Xu04c: Xu MG, Li J, Du X, Wang ED (2004). "Groups on the side chain of T252 in Escherichia coli leucyl-tRNA synthetase are important for discrimination of amino acids and cell viability." Biochem Biophys Res Commun 318(1);11-6. PMID: 15110746
Zhai07: Zhai Y, Nawaz MH, Lee KW, Kirkbride E, Briggs JM, Martinis SA (2007). "Modulation of substrate specificity within the amino acid editing site of leucyl-tRNA synthetase." Biochemistry 46(11);3331-7. PMID: 17311409
Zhou11: Zhou XL, Du DH, Tan M, Lei HY, Ruan LL, Eriani G, Wang ED (2011). "Role of tRNA amino acid-accepting end in aminoacylation and its quality control." Nucleic Acids Res 39(20);8857-68. PMID: 21775341
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