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MetaCyc Enzyme: glutathione synthetase

Gene: gshB Accession Numbers: EG10419 (MetaCyc), b2947, ECK2942

Synonyms: gsh-II

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of glutathione synthetase = [GshB]4
         glutathione synthetase monomer = GshB

Summary:
Glutathione synthetase catalyzes the second, final step in the pathway for the biosynthesis of glutathione [Apontoweil75].

Crystal structures of the enzyme have been solved [Kato89, Yamaguchi93, Hara96a, Matsuda96]. Site-directed mutagenesis identified the R233 residue [Tanaka92] and the importance of flexibility of the I226-G242 loop [Tanaka93] for catalytic activity. The loop is thought to enhance the recognition of glycine and stabilize the reaction intermediate [Fan95, Tanaka97]. In the crystal structure of the quarternary complex with ADP, glutathione and sulfate, the flexible loops are fixed, allowing better understanding of the active site and the mechanism of substrate recognition [Hara96a]. Further site-directed mutagenesis was used to probe the γ-glutamyl cysteine binding site [Hara95].

A strain overexpressing GshA and GshB is more resistant to radiation than wild type [Moore89]. In cells lacking metal efflux systems, glutathione biosynthesis is required for resistance to Cd2+, Zn2+ and Cu2+ [Helbig08]. A gshB trxB double mutant exhibits a growth defect which can be suppressed by growth in rich medium containing DTT or by gain-of-function mutations in ahpC [Faulkner08].

Transcription of gshB increases with increased cadmium concentration. A microarray analysis measuring the transcriptional response of wild-type and gshB mutant strains to cadmium stress has been performed [Helbig08a].

Citations: [Daws89, Hibi96]

Locations: cytosol

Map Position: [3,089,900 -> 3,090,850]

Molecular Weight of Polypeptide: 35.561 kD (from nucleotide sequence), 38.0 kD (experimental) [Gushima83 ]

Molecular Weight of Multimer: 152.0 kD (experimental) [Gushima83]

pI: 5.31

Unification Links: ASAP:ABE-0009667 , CGSC:33370 , DIP:DIP-9840N , EchoBASE:EB0414 , EcoGene:EG10419 , EcoliWiki:b2947 , ModBase:P04425 , OU-Microarray:b2947 , PortEco:gshB , Pride:P04425 , Protein Model Portal:P04425 , RefSeq:NP_417422 , RegulonDB:EG10419 , SMR:P04425 , String:511145.b2947 , UniProt:P04425

Relationship Links: InterPro:IN-FAMILY:IPR004215 , InterPro:IN-FAMILY:IPR004218 , InterPro:IN-FAMILY:IPR006284 , InterPro:IN-FAMILY:IPR011761 , InterPro:IN-FAMILY:IPR013815 , InterPro:IN-FAMILY:IPR013816 , InterPro:IN-FAMILY:IPR016185 , PDB:Structure:1GLV , PDB:Structure:1GSA , PDB:Structure:1GSH , PDB:Structure:2GLT , Pfam:IN-FAMILY:PF02951 , Pfam:IN-FAMILY:PF02955 , Prosite:IN-FAMILY:PS50975

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006750 - glutathione biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, UniProtGOA11, GOA06, GOA01, Fuchs75, Apontoweil75a]
Molecular Function: GO:0000287 - magnesium ion binding Inferred from experiment Inferred by computational analysis [GOA06, Gushima83]
GO:0004363 - glutathione synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01a, GOA01, Gushima83]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0003824 - catalytic activity Inferred by computational analysis [GOA01]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11, GOA06, GOA01]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
GO:0030145 - manganese ion binding Inferred by computational analysis [GOA06]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11, GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [GOA01, DiazMejia09, Ishihama08, LopezCampistrou05, Lasserre06]

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

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International


Enzymatic reaction of: glutathione synthetase

Synonyms: GSH synthetase, γ-L-glutamyl-L-cysteine:glycine ligase (ADP-forming)

EC Number: 6.3.2.3

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

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for glycine [Comment 1 ]: glycine ethyl ester [Kato94 ] , β-alanine [Kato94 ] , glycine methyl ester [Kato94 ]

In Pathways: glutathione biosynthesis

Credits:
Imported from EcoCyc 16-Sep-2014 by Paley S , SRI International

Summary:
The enzyme was first purified from E. coli B [Gushima83, Kato88, Tanaka92]; the sequences of the B and K-12 genes appear to be identical.

Cofactors or Prosthetic Groups: Mg2+ [Comment 2, Gushima83]

Inhibitors (Unknown Mechanism): AP4-PL [Hibi93] , glutathione disulfide [Apontoweil75] , methotrexate [Kato87] , p-chloromercuribenzoate [Kato88] , 5,5'-dithio-bis-2-nitrobenzoate [Kato88]

Kinetic Parameters:

Substrate
Km (μM)
Citations
ATP
240.0
[Tanaka97, BRENDA14]
ATP
1850.0
[Kato87, BRENDA14]
ATP
1800.0
[Gushima83, BRENDA14]
glycine
910.0
[Tanaka97, BRENDA14]
glycine
2000.0
[Gushima83, BRENDA14]
glycine
3530.0
[Kato87, BRENDA14]
glycine
400.0
[Apontoweil75, BRENDA14]
γ-L-glutamyl-L-cysteine
630.0
[Apontoweil75]

T(opt): 45 °C [Gushima83]

pH(opt): 7.5 [BRENDA14, Matsuda96], 8 [BRENDA14, Gushima83], 8.5 [BRENDA14, Apontoweil75], 8-9 [Apontoweil75]


Sequence Features

Feature Class Location Citations Comment
Mutagenesis-Variant 122
[Kato88, UniProt11a]
Alternate sequence: A; UniProt: No loss of activity.
Conserved-Region 125 -> 310
[UniProt09]
UniProt: ATP-grasp;
Nucleotide-Phosphate-Binding-Region 151 -> 207
[UniProt10]
UniProt: ATP; Non-Experimental Qualifier: by similarity;
Mutagenesis-Variant 195
[Kato88, UniProt11a]
Alternate sequence: A; UniProt: No loss of activity.
Mutagenesis-Variant 222
[Kato88, UniProt11a]
Alternate sequence: A; UniProt: No loss of activity.
Mutagenesis-Variant 227
[Kato88, Tanaka93, UniProt11a]
Alternate sequence: V; UniProt: Loss of activity.
Mutagenesis-Variant 233
[Kato88, Tanaka92, UniProt11a]
Alternate sequence: K; UniProt: Loss of activity.
Alternate sequence: A; UniProt: Loss of activity.
Mutagenesis-Variant 240
[Kato88, Tanaka93, UniProt11a]
Alternate sequence: V; UniProt: Loss of activity.
Mutagenesis-Variant 241
[Kato88, Tanaka92, UniProt11a]
Alternate sequence: K; UniProt: No loss of activity.
Alternate sequence: A; UniProt: No loss of activity.
Metal-Binding-Site 281
[UniProt10]
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity;
Metal-Binding-Site 283
[UniProt10]
UniProt: Magnesium or manganese; Non-Experimental Qualifier: by similarity;
Mutagenesis-Variant 289
[Kato88, UniProt11a]
Alternate sequence: A; UniProt: No loss of activity.

History:
10/20/97 Gene b2947 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10419; confirmed by SwissProt match.


References

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

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014." http://www.brenda-enzymes.org.

Daws89: Daws T, Lim CJ, Fuchs JA (1989). "In vitro construction of gshB::kan in Escherichia coli and use of gshB::kan in mapping the gshB locus." J Bacteriol 171(9);5218-21. PMID: 2670910

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

Fan95: Fan C, Moews PC, Shi Y, Walsh CT, Knox JR (1995). "A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli." Proc Natl Acad Sci U S A 92(4);1172-6. PMID: 7862655

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

Fuchs75: Fuchs JA, Warner HR (1975). "Isolation of an Escherichia coli mutant deficient in glutathione synthesis." J Bacteriol 124(1);140-8. PMID: 1100598

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."

Gushima83: Gushima H, Miya T, Murata K, Kimura A (1983). "Purification and characterization of glutathione synthetase from Escherichia coli B." J Appl Biochem 1983;5(3);210-8. PMID: 6389479

Hara95: Hara T, Tanaka T, Kato H, Nishioka T, Oda J (1995). "Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site." Protein Eng 8(7);711-6. PMID: 8577699

Hara96a: Hara T, Kato H, Katsube Y, Oda J (1996). "A pseudo-michaelis quaternary complex in the reverse reaction of a ligase: structure of Escherichia coli B glutathione synthetase complexed with ADP, glutathione, and sulfate at 2.0 A resolution." Biochemistry 35(37);11967-74. PMID: 8810901

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

Hibi93: Hibi T, Kato H, Nishioka T, Oda J, Yamaguchi H, Katsube Y, Tanizawa K, Fukui T (1993). "Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B." Biochemistry 32(6);1548-54. PMID: 8431434

Hibi96: Hibi T, Nishioka T, Kato H, Tanizawa K, Fukui T, Katsube Y, Oda J (1996). "Structure of the multifunctional loops in the nonclassical ATP-binding fold of glutathione synthetase." Nat Struct Biol 3(1);16-8. PMID: 8548447

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

Kato87: Kato H, Chihara M, Nishioka T, Murata K, Kimura A, Oda J (1987). "Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site." J Biochem 101(1);207-15. PMID: 3553173

Kato88: Kato H, Tanaka T, Nishioka T, Kimura A, Oda J (1988). "Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis." J Biol Chem 1988;263(24);11646-51. PMID: 3042775

Kato89: Kato H, Yamaguchi H, Hata Y, Nishioka T, Katsube Y, Oda J (1989). "Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B." J Mol Biol 209(3);503-4. PMID: 2685323

Kato94: Kato H, Tanaka T, Yamaguchi H, Hara T, Nishioka T, Katsube Y, Oda J (1994). "Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase." Biochemistry 33(17);4995-9. PMID: 8172874

Lasserre06: Lasserre JP, Beyne E, Pyndiah S, Lapaillerie D, Claverol S, Bonneu M (2006). "A complexomic study of Escherichia coli using two-dimensional blue native/SDS polyacrylamide gel electrophoresis." Electrophoresis 27(16);3306-21. PMID: 16858726

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Matsuda96: Matsuda K, Mizuguchi K, Nishioka T, Kato H, Go N, Oda J (1996). "Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins." Protein Eng 9(12);1083-92. PMID: 9010922

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

Tanaka92: Tanaka T, Kato H, Nishioka T, Oda J (1992). "Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction." Biochemistry 1992;31(8);2259-65. PMID: 1540581

Tanaka93: Tanaka T, Yamaguchi H, Kato H, Nishioka T, Katsube Y, Oda J (1993). "Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase." Biochemistry 1993;32(46);12398-404. PMID: 8241129

Tanaka97: Tanaka T, Nishioka T, Oda J (1997). "Nicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate." Arch Biochem Biophys 339(1);151-6. PMID: 9056244

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt11a: UniProt Consortium (2011). "UniProt version 2011-06 released on 2011-06-30 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."

Yamaguchi93: Yamaguchi H, Kato H, Hata Y, Nishioka T, Kimura A, Oda J, Katsube Y (1993). "Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution." J Mol Biol 1993;229(4);1083-100. PMID: 8445637


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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Fri Nov 28, 2014, biocyc13.