Escherichia coli K-12 substr. MG1655 Enzyme: leucyl-tRNA synthetase

Gene: leuS Accession Numbers: EG10532 (EcoCyc), b0642, ECK0635

Synonyms: syl

Regulation Summary Diagram: ?

Regulation summary diagram for leuS

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 [Chen00, Mursinna01, Du02]. The Thr251 residue is a critical specificity determinant [Mursinna04, Xu04b], 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].

Reviews: [ODonoghue03, Yadavalli08]

Citations: [Brustad08, Tan12]

Locations: cytosol

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)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0006429 - leucyl-tRNA aminoacylation Inferred from experiment Inferred by computational analysis [GOA06, GOA01, Chen99]
GO:0006412 - translation Inferred by computational analysis [UniProtGOA11]
GO:0006418 - tRNA aminoacylation for protein translation Inferred by computational analysis [GOA01]
GO:0006450 - regulation of translational fidelity Inferred by computational analysis [GOA01]
Molecular Function: GO:0004823 - leucine-tRNA ligase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Chen99]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0002161 - aminoacyl-tRNA editing activity Inferred by computational analysis [GOA01]
GO:0004812 - aminoacyl-tRNA ligase activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11, GOA06, GOA01]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [Gaudet10, DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, GOA06]

MultiFun Terms: information transfer protein related amino acid -activation

Essentiality data for leuS knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox No 37 Aerobic 7   No [Baba06, Comment 1]

Last-Curated ? 27-Jun-2012 by Keseler I , SRI International

Enzymatic reaction of: leucyl-tRNA synthetase

Synonyms: LeuRS

EC Number:

a tRNAleu + L-leucine + ATP + H+ <=> an L-leucyl-[tRNAleu] + AMP + diphosphate

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

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
a tRNAleu
[Xu04b, BRENDA14]
a tRNAleu
[Zhai07, BRENDA14]
a tRNAleu
[Chen00, BRENDA14]
a tRNAleu
1.6, 1.6
3.4, 3.9
2.13, 2.44, 2.12, 2.44
[Chen99, BRENDA14]
a tRNAleu
[Du03, BRENDA14]
a tRNAleu
[Chen01c, BRENDA14]
a tRNAleu
[Vu07, BRENDA14]
[Xu04b, BRENDA14]
[Chen99, BRENDA14]
[Chen01c, BRENDA14]
[Mursinna04, BRENDA14]
[Lue05, BRENDA14]
[Chen00, BRENDA14]
[Xu04b, BRENDA14]
280.0, 260.0
3.6, 4.2
0.013, 0.015, 0.014, 0.016
[Chen99, BRENDA14]
[Du03, BRENDA14]

Sequence Features

Protein sequence of leucyl-tRNA synthetase with features indicated

Feature Class Location Citations Comment
Protein-Segment 42 -> 52
UniProt: "HIGH" region; Sequence Annotation Type: short sequence motif.
Sequence-Conflict 67
[Hartlein87, UniProt10]
UniProt: (in Ref. 1; CAA29642);
Sequence-Conflict 196
[Hartlein87, UniProt10]
UniProt: (in Ref. 1; CAA29642);
Sequence-Conflict 262
[Chung97, Hartlein87, UniProt10]
UniProt: (in Ref. 1 and 3);
Protein-Segment 619 -> 623
UniProt: "KMSKS" region; Sequence Annotation Type: short sequence motif.
Amino-Acid-Sites-That-Bind 622
UniProt: ATP; Non-Experimental Qualifier: by similarity;

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


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

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014."

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

Chen00: Chen JF, Guo NN, Li T, Wang ED, Wang YL (2000). "CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function." Biochemistry 39(22);6726-31. PMID: 10828991

Chen01c: 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

Chen99: Chen J, Li Y, Wang E, Wang Y (1999). "High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli." Protein Expr Purif 15(1);115-20. PMID: 10024478

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

Du02: Du X, Wang ED (2002). "Discrimination of tRNA(Leu) isoacceptors by the mutants of Escherichia coli leucyl-tRNA synthetase in editing." Biochemistry 41(34);10623-8. PMID: 12186547

Du03: Du X, Wang ED (2003). "E292 is important for the aminoacylation activity of Escherichia coli leucyl-tRNA synthetase." J Protein Chem 22(1);71-6. PMID: 12739900

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

Gaudet10: Gaudet P, Livstone M, Thomas P (2010). "Annotation inferences using phylogenetic trees." PMID: 19578431

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Hartlein87: Hartlein M, Madern D (1987). "Molecular cloning and nucleotide sequence of the gene for Escherichia coli leucyl-tRNA synthetase." Nucleic Acids Res 15(24);10199-210. PMID: 3320963

Hellmann09: Hellmann RA, Martinis SA (2009). "Defects in transient tRNA translocation bypass tRNA synthetase quality control mechanisms." J Biol Chem 284(17);11478-84. PMID: 19258309

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

Hsu08: Hsu JL, Martinis SA (2008). "A Flexible peptide tether controls accessibility of a unique C-terminal RNA-binding domain in leucyl-tRNA synthetases." J Mol Biol 376(2);482-91. PMID: 18155724

Ibba00: Ibba M, Soll D (2000). "Aminoacyl-tRNA synthesis." Annu Rev Biochem 69;617-50. PMID: 10966471

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

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

Lue05: Lue SW, Kelley SO (2005). "An aminoacyl-tRNA synthetase with a defunct editing site." Biochemistry 44(8);3010-6. PMID: 15723544

Lue07: Lue SW, Kelley SO (2007). "A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation." Biochemistry 46(15);4466-72. PMID: 17378584

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

ODonoghue03: O'Donoghue P, Luthey-Schulten Z (2003). "On the evolution of structure in aminoacyl-tRNA synthetases." Microbiol Mol Biol Rev 67(4);550-73. PMID: 14665676

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

Starzyk87: Starzyk RM, Webster TA, Schimmel P (1987). "Evidence for dispensable sequences inserted into a nucleotide fold." Science 237(4822);1614-8. PMID: 3306924

Tan10: Tan M, Zhu B, Zhou XL, He R, Chen X, Eriani G, Wang ED (2010). "tRNA-dependent pre-transfer editing by prokaryotic leucyl-tRNA synthetase." J Biol Chem 285(5);3235-44. PMID: 19940155

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

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

Vu07: Vu MT, Martinis SA (2007). "A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing." Biochemistry 46(17);5170-6. PMID: 17407263

Williams06: Williams AM, Martinis SA (2006). "Mutational unmasking of a tRNA-dependent pathway for preventing genetic code ambiguity." Proc Natl Acad Sci U S A 103(10);3586-91. PMID: 16505383

Xu04b: 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

Yadavalli08: Yadavalli SS, Musier-Forsyth K, Ibba M (2008). "The return of pretransfer editing in protein synthesis." Proc Natl Acad Sci U S A 105(49);19031-2. PMID: 19057010

Zhai05: Zhai Y, Martinis SA (2005). "Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism." Biochemistry 44(47);15437-43. PMID: 16300391

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

Zhou08a: Zhou XL, Zhu B, Wang ED (2008). "The CP2 domain of leucyl-tRNA synthetase is crucial for amino acid activation and post-transfer editing." J Biol Chem 283(52);36608-16. PMID: 18955487

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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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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