Escherichia coli K-12 substr. MG1655 Enzyme: fused glutathionylspermidine amidase / glutathionylspermidine synthetase

Gene: gss Accession Numbers: EG12882 (EcoCyc), b2988, ECK2982

Synonyms: gsp

Regulation Summary Diagram: ?

Regulation summary diagram for gss

Subunit composition of fused glutathionylspermidine amidase / glutathionylspermidine synthetase = [Gss]2

gss encodes a bifunctional protein with two domains: an N-terminal glutathionylspermidine (GSP) amidase domain and a C-terminal GSP synthetase domain. The enzyme catalyzes both the formation and hydrolysis of an amide bond between glutathione and spermidine. The net reaction of the two component activities is ATP hydrolysis, a potential futile cycle [Kwon97].

Glutathionylspermidine levels in E. coli have been measured; levels increase in stationary phase, especially under anaerobic conditions [Smith95] and after treatment with hydrogen peroxide [Chiang10]. The function of glutathionylspermidine metabolism in E. coli has been unclear. Recently it was proposed that the GSP amidase activity plays a role in reduction of Gsp-S-S-Gsp and Gsp-S-protein adducts [Chiang10].

Regulation of GSP amidase/synthetase at the enzyme level is required to avoid futile cycling. The two domains can be physically separated and fold and function independently. If expressed independently, the GSP amidase domain has significantly higher amidase activity than the full-length protein. Thus, in the context of the full-length protein, the amidase domain is negatively autoregulated [Kwon97]. However, amidase activity increases up to 15-fold by addition of synthetase substrates [Lin97]. Hydrogen peroxide selectively inactivates the amidase, but not the synthetase activity of Gss [Chiang10]; amidase activity recovers significantly after four hours [Chong11].

The catalytic mechanism for the amidase function is thought to involve a nucleophilic attack involving cysteine as the catalytic nucleophile and an acyl-enzyme intermediate. Cys59 was found to be essential for amidase activity [Lin97].

Various synthetic inhibitors have been designed and tested [Chen97, Lin97a].

Crystal structures of Gss alone or in complex with substrate, product or inhibitor have been solved; the structures are consistent with the proposed reaction mechanism [Pai06]. Crystal structures of the amidase domain alone, in complex with its substrate, and modified by hydrogen peroxide treatment have been solved [Chiang10, Pai11].

Gsp: "glutathionylspermidine" [Bollinger95]

Locations: cytosol

Map Position: [3,134,685 <- 3,136,544] (67.56 centisomes, 243°)
Length: 1860 bp / 619 aa

Molecular Weight of Polypeptide: 70.532 kD (from nucleotide sequence), 70 kD (experimental) [Bollinger95 ]

Molecular Weight of Multimer: 138 kD (experimental) [Pai06]

pI: 5.09 [Bollinger95]

Unification Links: ASAP:ABE-0009804 , DIP:DIP-36018N , EchoBASE:EB2720 , EcoGene:EG12882 , EcoliWiki:b2988 , Mint:MINT-1234290 , OU-Microarray:b2988 , PortEco:gsp , PR:PRO_000022849 , Pride:P0AES0 , Protein Model Portal:P0AES0 , RefSeq:NP_417462 , RegulonDB:EG12882 , SMR:P0AES0 , String:511145.b2988 , UniProt:P0AES0

Relationship Links: InterPro:IN-FAMILY:IPR005494 , InterPro:IN-FAMILY:IPR007921 , InterPro:IN-FAMILY:IPR016185 , PDB:Structure:2IO7 , PDB:Structure:2IO8 , PDB:Structure:2IO9 , PDB:Structure:2IOA , PDB:Structure:2IOB , PDB:Structure:3A2Y , PDB:Structure:3A2Z , PDB:Structure:3A30 , PDB:Structure:3O98 , Pfam:IN-FAMILY:PF03738 , Pfam:IN-FAMILY:PF05257 , Prosite:IN-FAMILY:PS50911

In Paralogous Gene Group: 477 (3 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for gss

GO Terms:

Biological Process: GO:0006749 - glutathione metabolic process Inferred by computational analysis [UniProtGOA12]
GO:0008152 - metabolic process Inferred by computational analysis [UniProtGOA11]
GO:0008216 - spermidine metabolic process Inferred by computational analysis [UniProtGOA12]
Molecular Function: GO:0005515 - protein binding Inferred from experiment [Rajagopala14, Butland05]
GO:0008884 - glutathionylspermidine amidase activity Inferred from experiment Inferred by computational analysis [GOA01a, Lin97]
GO:0008885 - glutathionylspermidine synthase activity Inferred from experiment Inferred by computational analysis [GOA01a, Lin97]
GO:0000166 - nucleotide binding Inferred by computational analysis [UniProtGOA11]
GO:0003824 - catalytic activity Inferred by computational analysis [UniProtGOA11]
GO:0005524 - ATP binding Inferred by computational analysis [UniProtGOA11]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]

MultiFun Terms: metabolism central intermediary metabolism polyamine biosynthesis

Essentiality data for gss 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 ? 23-May-2012 by Keseler I , SRI International

Enzymatic reaction of: glutathionylspermidine synthetase

Synonyms: GSP synthetase

EC Number:

spermidine + glutathione + ATP <=> glutathionylspermidine + 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 glutathione: γ-L-glutamyl-L-alanyl-glycine [Kwon97 ]

In Pathways: glutathionylspermidine biosynthesis

Cofactors or Prosthetic Groups: Mg2+ [Bollinger95]

Inhibitors (Unknown Mechanism): phosphonate [Kwon97]

Kinetic Parameters:

Km (μM)

pH(opt): 6.8 [Bollinger95]

Enzymatic reaction of: glutathionylspermidine amidase

Synonyms: GSP amidase

EC Number:

glutathionylspermidine + H2O <=> glutathione + spermidine

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 glutathionylspermidine: glutathionylspermidine disulfide [Chiang10 ]

Inhibitors (Unknown Mechanism): hydrogen peroxide [Chiang10] , γ-Glu-Ala-Gly-CHO [Lin97a] , iodoacetamide [Lin97]

Sequence Features

Protein sequence of Gss with features indicated

Feature Class Location Common Name Attached Group Citations Comment
Cleavage-of-Initial-Methionine 1    
[Bollinger95, UniProt15]
UniProt: Removed.
Protein-Segment 2 -> 195    
UniProt: Gsp amidase; Sequence Annotation Type: region of interest.
Chain 2 -> 619    
UniProt: Bifunctional glutathionylspermidine synthetase/amidase.
Conserved-Region 34 -> 176    
UniProt: Peptidase C51;
Amino-Acid-Sites-That-Bind 58    
UniProt: Gsp.
Mutagenesis-Variant 59    
[Lin97, UniProt15]
UniProt: Loss of amidase activity.
Cysteine-sulfenic-acid-Modification 59    
[Chiang10, UniProt15]
UniProt: Cysteine sulfenic acid (-SOH); transient.
Active-Site 59 catalytic nucleophile for amidase activity  
[Pai11, Chiang10, Lin97]
Amino-Acid-Sites-That-Bind 64    
UniProt: Gsp.
Protein-Segment 78 -> 81    
UniProt: Gsp binding; Sequence Annotation Type: region of interest.
Amino-Acid-Site 131    
UniProt: Increases nucleophilicity of active site Cys; for amidase activity; Sequence Annotation Type: site.
Amino-Acid-Sites-That-Bind 149    
UniProt: Gsp.
Mutagenesis-Variant 173    
[Lin97, UniProt15]
UniProt: No effect on amidase activity.
Protein-Segment 196 -> 205    
UniProt: Linker; Sequence Annotation Type: region of interest.
Protein-Segment 206 -> 619    
UniProt: Gsp synthetase; Sequence Annotation Type: region of interest.
Mutagenesis-Variant 316    
UniProt: Loss of synthetase activity.
Amino-Acid-Sites-That-Bind 316    
UniProt: Glutathione.
Nucleotide-Phosphate-Binding-Region 316 -> 318   ATP
UniProt: ATP.
Metal-Binding-Site 318    
UniProt: Magnesium 1.
Metal-Binding-Site 330    
UniProt: Magnesium 1.
Metal-Binding-Site 332    
UniProt: Magnesium 2.
Mutagenesis-Variant 335    
[Pai06, UniProt15]
UniProt: 3.6-fold decrease in GSH affinity, 1.6-fold decrease in spermidine activity, and 1.3-fold decrease in synthetase activity.
Amino-Acid-Sites-That-Bind 335    
UniProt: Glutathione.
Mutagenesis-Variant 337    
[Pai06, UniProt15]
UniProt: No effect on GSH and spermidine affinity, but 2-fold decrease in synthetase activity.
Mutagenesis-Variant 338    
[Pai06, UniProt15]
UniProt: 10-fold decrease in GSH affinity, 5-fold decrease in spermidine activity, but no effect on synthetase activity.
Mutagenesis-Variant 391    
[Pai06, UniProt15]
UniProt: 2-fold decrease in GSH affinity, 60-fold decrease in spermidine activity, and 10-fold decrease in synthetase activity.
Amino-Acid-Sites-That-Bind 391    
UniProt: Spermidine.
Mutagenesis-Variant 392    
[Pai06, UniProt15]
UniProt: 33-fold decrease in GSH affinity, 13-fold decrease in spermidine activity, and 6-fold decrease in synthetase activity.
Amino-Acid-Sites-That-Bind 392    
UniProt: Glutathione.
Mutagenesis-Variant 441    
[Pai06, UniProt15]
UniProt: 3-fold decrease in GSH affinity, 21-fold decrease in spermidine activity, and 17-fold decrease in synthetase activity.
Amino-Acid-Sites-That-Bind 446    
UniProt: Glutathione.
Amino-Acid-Sites-That-Bind 498    
UniProt: ATP.
Amino-Acid-Sites-That-Bind 533    
UniProt: ATP.
Mutagenesis-Variant 538    
[Pai06, UniProt15]
UniProt: 6-fold decrease in GSH affinity, 2.4-fold decrease in spermidine activity, and 4-fold decrease in synthetase activity.
Nucleotide-Phosphate-Binding-Region 539 -> 540   ATP
UniProt: ATP.
Nucleotide-Phosphate-Binding-Region 568 -> 571   ATP
UniProt: ATP.
Amino-Acid-Sites-That-Bind 582    
UniProt: ATP.
Mutagenesis-Variant 598    
[Pai06, UniProt15]
UniProt: 10-fold increase in GSH affinity, 9-fold decrease in spermidine activity, and 15-fold decrease in synthetase activity.
Nucleotide-Phosphate-Binding-Region 603 -> 605   ATP
UniProt: ATP.
Amino-Acid-Sites-That-Bind 610    
UniProt: Spermidine.

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2988 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG12882; 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

Bollinger95: Bollinger JM, Kwon DS, Huisman GW, Kolter R, Walsh CT (1995). "Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase." J Biol Chem 1995;270(23);14031-41. PMID: 7775463

Butland05: Butland G, Peregrin-Alvarez JM, Li J, Yang W, Yang X, Canadien V, Starostine A, Richards D, Beattie B, Krogan N, Davey M, Parkinson J, Greenblatt J, Emili A (2005). "Interaction network containing conserved and essential protein complexes in Escherichia coli." Nature 433(7025);531-7. PMID: 15690043

Chen97: Chen S, Lin CH, Kwon DS, Walsh CT, Coward JK (1997). "Design, synthesis, and biochemical evaluation of phosphonate and phosphonamidate analogs of glutathionylspermidine as inhibitors of glutathionylspermidine synthetase/amidase from Escherichia coli." J Med Chem 40(23);3842-50. PMID: 9371250

Chiang10: Chiang BY, Chen TC, Pai CH, Chou CC, Chen HH, Ko TP, Hsu WH, Chang CY, Wu WF, Wang AH, Lin CH (2010). "Protein S-thiolation by Glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetASE/amidase in redox regulation." J Biol Chem 285(33);25345-53. PMID: 20530482

Chong11: Chong CM, Gao S, Chiang BY, Hsu WH, Lin TC, Chen TC, Lin CH (2011). "An acyloxymethyl ketone-based probe to monitor the activity of glutathionylspermidine amidase in Escherichia coli." Chembiochem 12(15);2306-9. PMID: 21887840

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

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

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

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

Kwon97: Kwon DS, Lin CH, Chen S, Coward JK, Walsh CT, Bollinger JM (1997). "Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains." J Biol Chem 272(4);2429-36. PMID: 8999955

Lin97: Lin CH, Kwon DS, Bollinger JM, Walsh CT (1997). "Evidence for a glutathionyl-enzyme intermediate in the amidase activity of the bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli." Biochemistry 36(48);14930-8. PMID: 9398217

Lin97a: Lin CH, Chen S, Kwon DS, Coward JK, Walsh CT (1997). "Aldehyde and phosphinate analogs of glutathione and glutathionylspermidine: potent, selective binding inhibitors of the E. coli bifunctional glutathionylspermidine synthetase/amidase." Chem Biol 4(11);859-66. PMID: 9384533

Pai06: Pai CH, Chiang BY, Ko TP, Chou CC, Chong CM, Yen FJ, Chen S, Coward JK, Wang AH, Lin CH (2006). "Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase." EMBO J 25(24);5970-82. PMID: 17124497

Pai11: Pai CH, Wu HJ, Lin CH, Wang AH (2011). "Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases." Protein Sci 20(3);557-66. PMID: 21226054

Rajagopala14: Rajagopala SV, Sikorski P, Kumar A, Mosca R, Vlasblom J, Arnold R, Franca-Koh J, Pakala SB, Phanse S, Ceol A, Hauser R, Siszler G, Wuchty S, Emili A, Babu M, Aloy P, Pieper R, Uetz P (2014). "The binary protein-protein interaction landscape of Escherichia coli." Nat Biotechnol 32(3);285-90. PMID: 24561554

Smith95: Smith K, Borges A, Ariyanayagam MR, Fairlamb AH (1995). "Glutathionylspermidine metabolism in Escherichia coli." Biochem J 1995;312 ( Pt 2);465-9. PMID: 8526857

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

UniProt13: UniProt Consortium (2013). "UniProt version 2013-08 released on 2013-08-01 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."

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

Other References Related to Gene Regulation

Beisel11: Beisel CL, Storz G (2011). "The base-pairing RNA spot 42 participates in a multioutput feedforward loop to help enact catabolite repression in Escherichia coli." Mol Cell 41(3);286-97. PMID: 21292161

MendozaVargas09: Mendoza-Vargas A, Olvera L, Olvera M, Grande R, Vega-Alvarado L, Taboada B, Jimenez-Jacinto V, Salgado H, Juarez K, Contreras-Moreira B, Huerta AM, Collado-Vides J, Morett E (2009). "Genome-wide identification of transcription start sites, promoters and transcription factor binding sites in E. coli." PLoS One 4(10);e7526. PMID: 19838305

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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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