Escherichia coli K-12 substr. MG1655 Enzyme: γ-glutamate-cysteine ligase

Gene: gshA Accession Numbers: EG10418 (EcoCyc), b2688, ECK2683

Synonyms: gsh-I

Regulation Summary Diagram: ?

Regulation summary diagram for gshA

γ-Glutamate-cysteine ligase catalyzes the first of two steps in the pathway for the biosynthesis of glutathione. The enzyme is feedback-inhibited by glutathione [Apontoweil75].

The enzyme contains two divalent metal ions which play a role in amino acid binding. Substrate specificity depends on whether Mg2+ or Mn2+ is present; Mn2+ broadens the specificity [Kelly02]. Transition state analog inhibitors have been used to study substrate recognition [Hiratake02]. The enzyme can utilize ethylamine as a substrate, thus producing theanine (γ-glutamylethylamide) [Miyake09].

Crystal structures of the enzyme from E. coli B (whose wild-type sequence is identical to the K-12 enzyme) have been solved [Hibi02, Hibi04]. The structure shows coordination of an inhibitor by three Mg2+ ions, and showing that the binding of cysteine follows an induced-fit model [Hibi04]. Although not highly conserved, the cysteine residues C372 and C395 of the intrachain disulfide bond are important for catalytic activity and stability of the enzyme [Kumar13].

A gshA insertion mutant is more sensitive to thiol-specific damage [Greenberg86], mercury and arsenite [Latinwo98], and is unable to grow at high osmolarity [McLaggan90], but is not affected in its resistance to oxidative damage [Greenberg86]. However, a strain overexpressing GshA and GshB is more resistant to radiation than wild type [Moore89]. A gshA trxB double mutant exhibits a severe growth defect which can be suppressed by growth in rich medium containing DTT or by certain mutations in aphC [Faulkner08]. In cells lacking metal efflux systems, glutathione biosynthesis is required for resistance to Cd2+, Zn2+ and Cu2+ [Helbig08]. The resistance of a gshA mutant to a variety of metals has been measured [Harrison09]. As shown by the effect of a gshA mutation, glutathione plays a role in formation of the active, [4Fe-4S] cluster-containing form of FNR [Tran00].

Suppressor mutations that allow a ΔgshA strain to grow in the presence of arsenate map to proA and proB; enabling synthesis of glutathione [Veeravalli11].

Transcription of gshA increases with increased cadmium concentration. A microarray analysis measuring the transcriptional response of wild-type and gshA mutant strains to cadmium stress has been performed [Helbig08a]. Under oxidative stress conditions, translation of gshA is increased by the presence of polyamines. The increase is dependent on the non-canonical UUG start codon of the gshA open reading frame [Sakamoto15].

gshA was first cloned and sequenced from E. coli B [Watanabe86]; the sequence of the E. coli B and K-12 genes appear to be identical.

Citations: [Smirnova01]

Locations: cytosol

Map Position: [2,812,905 <- 2,814,461] (60.63 centisomes, 218°)
Length: 1557 bp / 518 aa

Molecular Weight of Polypeptide: 58.269 kD (from nucleotide sequence), 56.0 kD (experimental) [Huang88 ]

pI: 5.47

Unification Links: ASAP:ABE-0008845 , CGSC:659 , DIP:DIP-48212N , EchoBASE:EB0413 , EcoGene:EG10418 , EcoliWiki:b2688 , OU-Microarray:b2688 , PortEco:gshA , PR:PRO_000022843 , Pride:P0A6W9 , Protein Model Portal:P0A6W9 , RefSeq:NP_417173 , RegulonDB:EG10418 , SMR:P0A6W9 , String:511145.b2688 , UniProt:P0A6W9

Relationship Links: InterPro:IN-FAMILY:IPR006334 , InterPro:IN-FAMILY:IPR007370 , PDB:Structure:1V4G , PDB:Structure:1VA6 , PDB:Structure:2D32 , PDB:Structure:2D33 , Pfam:IN-FAMILY:PF04262

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for gshA

GO Terms:

Biological Process: GO:0006750 - glutathione biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA06, GOA01, Apontoweil75a]
GO:0006972 - hyperosmotic response Inferred from experiment [McLaggan90]
GO:0071243 - cellular response to arsenic-containing substance Inferred from experiment [Latinwo98]
GO:0071288 - cellular response to mercury ion Inferred from experiment [Latinwo98]
Molecular Function: GO:0004357 - glutamate-cysteine ligase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Huang88]
GO:0046872 - metal ion binding Inferred from experiment [Kelly02]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: metabolism biosynthesis of building blocks cofactors, small molecule carriers glutathione

Essentiality data for gshA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Last-Curated ? 06-May-2015 by Keseler I , SRI International

Enzymatic reaction of: glutamate-cysteine ligase

Synonyms: γ-glutamylcysteine synthetase, L-glutamate:L-cysteine γ-ligase (ADP-forming), γGCS

EC Number:

L-cysteine + L-glutamate + ATP <=> γ-L-glutamyl-L-cysteine + ADP + phosphate + H+

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.

Alternative Substrates for L-cysteine: (S)-2-aminobutanoate [Kelly02 ] , β-cyano-L-alanine [Kelly02 ] , S-methyl-L-cysteine [Kelly02 ]

Alternative Substrates for L-glutamate: D-glutamate [Kelly02 ] , N-methyl-L-glutamate [Kelly02 ] , α-ethyl-L-glutamate [Kelly02 ]

In Pathways: glutathione biosynthesis

Transition state analogues have been tested as inhibitors of the enzyme [Katoh98, Tokutake98, Hiratake02]. Inhibition by buthionine sulfoximine is dependent on the presence of ATP and is relatively weak [Kelly02].

Cofactors or Prosthetic Groups: Mg2+ [Kelly02]

Inhibitors (Irreversible): buthionine sulfoximine [Huang88]

Inhibitors (Unknown Mechanism): glutathione [Apontoweil75]

Primary Physiological Regulators of Enzyme Activity: glutathione

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)

pH(opt): 8-9 [Apontoweil75]

Sequence Features

Protein sequence of gamma-glutamate-cysteine ligase with features indicated

Feature Class Location Citations Comment State
Sequence-Conflict 100
[Watanabe86, UniProt10a]
UniProt: (in Ref. 1; CAA27583);
Active-Site 150
Intrachain-Disulfide-Bond 372, 395
[Hibi04, Hibi04]
Sequence-Conflict 494 -> 495
[Watanabe86, UniProt10a]
UniProt: (in Ref. 1; CAA27583);

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2688 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10418; confirmed by SwissProt match.


Apontoweil75: Apontoweil P, Berends W (1975). "Glutathione biosynthesis in Escherichia coli K 12. Properties of the enzymes and regulation." Biochim Biophys Acta 1975;399(1);1-9. PMID: 238647

Apontoweil75a: Apontoweil P, Berends W (1975). "Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12." Biochim Biophys Acta 399(1);10-22. PMID: 1096956

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

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

Faulkner08: Faulkner MJ, Veeravalli K, Gon S, Georgiou G, Beckwith J (2008). "Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways." Proc Natl Acad Sci U S A 105(18);6735-40. PMID: 18456836

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

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."

Greenberg86: Greenberg JT, Demple B (1986). "Glutathione in Escherichia coli is dispensable for resistance to H2O2 and gamma radiation." J Bacteriol 168(2);1026-9. PMID: 3536846

Harrison09: Harrison JJ, Tremaroli V, Stan MA, Chan CS, Vacchi-Suzzi C, Heyne BJ, Parsek MR, Ceri H, Turner RJ (2009). "Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance." Environ Microbiol 11(10);2491-509. PMID: 19555372

Helbig08: Helbig K, Bleuel C, Krauss GJ, Nies DH (2008). "Glutathione and transition-metal homeostasis in Escherichia coli." J Bacteriol 190(15);5431-8. PMID: 18539744

Helbig08a: Helbig K, Grosse C, Nies DH (2008). "Cadmium toxicity in glutathione mutants of Escherichia coli." J Bacteriol 190(15);5439-54. PMID: 18539742

Hibi02: Hibi T, Hisada H, Nakatsu T, Kato H, Oda J (2002). "Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis." Acta Crystallogr D Biol Crystallogr 58(Pt 2);316-8. PMID: 11807262

Hibi04: Hibi T, Nii H, Nakatsu T, Kimura A, Kato H, Hiratake J, Oda J (2004). "Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis." Proc Natl Acad Sci U S A 101(42);15052-7. PMID: 15477603

Hiratake02: Hiratake J, Irie T, Tokutake N, Oda J (2002). "Recognition of a cysteine substrate by E. coli gamma-glutamylcysteine synthetase probed by sulfoximine-based transition-state analogue inhibitors." Biosci Biotechnol Biochem 66(7);1500-14. PMID: 12224634

Huang88: Huang CS, Moore WR, Meister A (1988). "On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation." Proc Natl Acad Sci U S A 85(8);2464-8. PMID: 2895925

Inoue93: Inoue Y, Iba Y, Yano H, Murata K, Kimura A (1993). "Functional analysis of the gamma-glutamylcysteine synthetase of Escherichia coli B: effect of substitution of His-150 to Ala." Appl Microbiol Biotechnol 38(4);473-7. PMID: 7765288

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

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Katoh98: Katoh M, Hiratake J, Oda J (1998). "ATP-dependent inactivation of Escherichia coli gamma-glutamylcysteine synthetase by L-glutamic acid gamma-monohydroxamate." Biosci Biotechnol Biochem 62(7);1455-7. PMID: 9720231

Kelly02: Kelly BS, Antholine WE, Griffith OW (2002). "Escherichia coli gamma-glutamylcysteine synthetase. Two active site metal ions affect substrate and inhibitor binding." J Biol Chem 277(1);50-8. PMID: 11675389

Kumar13: Kumar S, Kasturia N, Sharma A, Datt M, Bachhawat AK (2013). "Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation." Biochem J 449(3);783-94. PMID: 23126248

Latinwo98: Latinwo LM, Donald C, Ikediobi C, Silver S (1998). "Effects of intracellular glutathione on sensitivity of Escherichia coli to mercury and arsenite." Biochem Biophys Res Commun 242(1);67-70. PMID: 9439611

McLaggan90: McLaggan D, Logan TM, Lynn DG, Epstein W (1990). "Involvement of gamma-glutamyl peptides in osmoadaptation of Escherichia coli." J Bacteriol 172(7);3631-6. PMID: 1972940

Miyake09: Miyake K, Kakita S (2009). "A novel catalytic ability of gamma-glutamylcysteine synthetase of Escherichia coli and its application in theanine production." Biosci Biotechnol Biochem 73(12);2677-83. PMID: 19966457

Moore89: Moore WR, Anderson ME, Meister A, Murata K, Kimura A (1989). "Increased capacity for glutathione synthesis enhances resistance to radiation in Escherichia coli: a possible model for mammalian cell protection." Proc Natl Acad Sci U S A 86(5);1461-4. PMID: 2564202

Sakamoto15: Sakamoto A, Terui Y, Yoshida T, Yamamoto T, Suzuki H, Yamamoto K, Ishihama A, Igarashi K, Kashiwagi K (2015). "Three Members of Polyamine Modulon under Oxidative Stress Conditions: Two Transcription Factors (SoxR and EmrR) and a Glutathione Synthetic Enzyme (GshA)." PLoS One 10(4);e0124883. PMID: 25898225

Smirnova01: Smirnova GV, Krasnykh TA, Oktyabrsky ON (2001). "Role of glutathione in the response of Escherichia coli to osmotic stress." Biochemistry (Mosc) 66(9);973-8. PMID: 11703177

Tokutake98: Tokutake N, Hiratake J, Katoh M, Irie T, Kato H, Oda J (1998). "Design, synthesis and evaluation of transition-state analogue inhibitors of Escherichia coli gamma-glutamylcysteine synthetase." Bioorg Med Chem 6(10);1935-53. PMID: 9839023

Tran00: Tran QH, Arras T, Becker S, Holighaus G, Ohlberger G, Unden G (2000). "Role of glutathione in the formation of the active form of the oxygen sensor FNR ([4Fe-4S].FNR) and in the control of FNR function." Eur J Biochem 267(15);4817-24. PMID: 10903516

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

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

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Veeravalli11: Veeravalli K, Boyd D, Iverson BL, Beckwith J, Georgiou G (2011). "Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways." Nat Chem Biol 7(2);101-5. PMID: 21186348

Watanabe86: Watanabe K, Yamano Y, Murata K, Kimura A (1986). "The nucleotide sequence of the gene for gamma-glutamylcysteine synthetase of Escherichia coli." Nucleic Acids Res 14(11);4393-400. PMID: 2872655

Other References Related to Gene Regulation

Maciag11: Maciag A, Peano C, Pietrelli A, Egli T, De Bellis G, Landini P (2011). "In vitro transcription profiling of the {sigma}S subunit of bacterial RNA polymerase: re-definition of the {sigma}S regulon and identification of {sigma}S-specific promoter sequence elements." Nucleic Acids Res 39(13);5338-55. PMID: 21398637

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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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