Skip Header

You are using a version of browser that may not display all the features of this website. Please consider upgrading your browser.
Protein

Bifunctional glutathionylspermidine synthetase/amidase

Gene

gss

Organism
Escherichia coli (strain K12)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Catalyzes the formation of an amide bond between glutathione (GSH) and spermidine coupled with hydrolysis of ATP; also catalyzes the opposing reaction, i.e. the hydrolysis of glutathionylspermidine (Gsp) back to glutathione and spermidine. The amidase active site can also hydrolyze Gsp-disulfide (Gsp-S-S-Gsp) to Gsp-SG and Gsp S-thiolated proteins (GspSSPs) to GSH S-thiolated protein (GSSPs). Likely acts synergistically with glutaredoxin to regulate the redox environment of E.coli and defend against oxidative damage. In vitro, the amidase active site also catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1).4 Publications

Catalytic activityi

Glutathione + spermidine + ATP = glutathionylspermidine + ADP + phosphate.4 Publications
Glutathionylspermidine + H2O = glutathione + spermidine.4 Publications

Enzyme regulationi

When exposed to oxidative stress, Gsp amidase activity is transiently inhibited in vivo by oxidation of the catalytic Cys-59 thiol to sulfenic acid; this modification does not affect Gsp synthetase activity. Gsp amidase activity is negatively autoregulated by the Gsp synthetase domain, and is activated by the Gsp synthetase substrates, GSH and ATP-Mg2+; the occupancy of the synthetase active site may initiate communication through the protein as manifest by the release of inhibition of the amidase activity. A tetrahedral phosphonate analog of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki of 10 µM) but does not inhibit the amidase activity. Amidase activity is inhibited by iodoacetamide in vitro.3 Publications

Kineticsi

kcat is 7 sec(-1) for Gsp synthetase activity at pH 6.8 and 2.1 sec(-1) for Gsp amidase activity at pH 7.5.1 Publication

Manual assertion based on experiment ini

  1. KM=100 µM for ATP (at pH 6.8)2 Publications
  2. KM=800 µM for glutathione (at pH 6.8)2 Publications
  3. KM=218 µM for glutathione2 Publications
  4. KM=60 µM for spermidine (at pH 6.8)2 Publications
  5. KM=20 µM for spermidine (at pH 7.5)2 Publications
  6. KM=76 µM for spermidine2 Publications
  7. KM=900 µM for glutathionylspermidine (at pH 7.5)2 Publications

    pH dependencei

    Optimum pH is around 6.8 for Gsp synthetase activity.1 Publication

    Pathwayi: glutathione metabolism

    This protein is involved in the pathway glutathione metabolism, which is part of Sulfur metabolism.
    View all proteins of this organism that are known to be involved in the pathway glutathione metabolism and in Sulfur metabolism.

    Pathwayi: spermidine metabolism

    This protein is involved in the pathway spermidine metabolism, which is part of Amine and polyamine metabolism.
    View all proteins of this organism that are known to be involved in the pathway spermidine metabolism and in Amine and polyamine metabolism.

    Sites

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Binding sitei58Gsp1
    Active sitei59S-(gamma-glutamyl-cysteinyl-glycyl)-cysteine intermediate1 Publication1
    Binding sitei64Gsp1
    Sitei131Increases nucleophilicity of active site Cys; for amidase activity1
    Binding sitei149Gsp1
    Binding sitei316Glutathione1
    Sitei316Transition state stabilizer; for synthetase activity1
    Metal bindingi318Magnesium 11
    Metal bindingi330Magnesium 11
    Metal bindingi330Magnesium 21
    Metal bindingi332Magnesium 21
    Binding sitei335Glutathione1
    Binding sitei391Spermidine1
    Binding sitei392Glutathione1
    Binding sitei446Glutathione1
    Binding sitei498ATP1
    Binding sitei533ATP1
    Binding sitei582ATP1
    Binding sitei610Spermidine1

    Regions

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Nucleotide bindingi316 – 318ATP3
    Nucleotide bindingi539 – 540ATP2
    Nucleotide bindingi568 – 571ATP4
    Nucleotide bindingi603 – 605ATP3

    GO - Molecular functioni

    • ATP binding Source: UniProtKB-KW
    • glutathionylspermidine amidase activity Source: EcoCyc
    • glutathionylspermidine synthase activity Source: EcoCyc
    • metal ion binding Source: UniProtKB-KW

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Hydrolase, Ligase

    Keywords - Ligandi

    ATP-binding, Magnesium, Metal-binding, Nucleotide-binding

    Enzyme and pathway databases

    BioCyciEcoCyc:GSP-MONOMER.
    ECOL316407:JW2956-MONOMER.
    MetaCyc:GSP-MONOMER.
    BRENDAi3.5.1.78. 2026.
    6.3.1.8. 2026.
    UniPathwayiUPA00204.
    UPA00819.

    Protein family/group databases

    MEROPSiC51.A01.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    Bifunctional glutathionylspermidine synthetase/amidase
    Short name:
    GspSA
    Including the following 2 domains:
    Glutathionylspermidine amidase (EC:3.5.1.784 Publications)
    Short name:
    Gsp amidase
    Alternative name(s):
    Glutathionylspermidine amidohydrolase [spermidine-forming]
    Glutathionylspermidine synthetase (EC:6.3.1.84 Publications)
    Short name:
    Gsp synthetase
    Alternative name(s):
    Glutathione:spermidine ligase [ADP-forming]
    Gsp synthase
    Gene namesi
    Name:gss
    Synonyms:gsp
    Ordered Locus Names:b2988, JW2956
    OrganismiEscherichia coli (strain K12)
    Taxonomic identifieri83333 [NCBI]
    Taxonomic lineageiBacteriaProteobacteriaGammaproteobacteriaEnterobacteralesEnterobacteriaceaeEscherichia
    Proteomesi
    • UP000000318 Componenti: Chromosome
    • UP000000625 Componenti: Chromosome

    Organism-specific databases

    EcoGeneiEG12882. gss.

    Subcellular locationi

    GO - Cellular componenti

    • cytosol Source: EcoCyc
    Complete GO annotation...

    Pathology & Biotechi

    Disruption phenotypei

    Cells lacking this gene do not produce Gsp under anaerobic conditions. Cells lacking both this gene and glutaredoxin (grxA or grxB) become hypersensitive to H2O2; they are even more susceptible to oxidative damage than the single mutant lacking glutaredoxin only.2 Publications

    Mutagenesis

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Mutagenesisi59C → A: Loss of amidase activity. 1 Publication1
    Mutagenesisi173C → A: No effect on amidase activity. 1 Publication1
    Mutagenesisi316R → E: Loss of synthetase activity. 1
    Mutagenesisi335S → A: 3.6-fold decrease in GSH affinity, 1.6-fold decrease in spermidine activity, and 1.3-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi337S → A: No effect on GSH and spermidine affinity, but 2-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi338C → A: 10-fold decrease in GSH affinity, 5-fold decrease in spermidine activity, but no effect on synthetase activity. 1 Publication1
    Mutagenesisi391E → A: 2-fold decrease in GSH affinity, 60-fold decrease in spermidine activity, and 10-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi392E → A: 33-fold decrease in GSH affinity, 13-fold decrease in spermidine activity, and 6-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi441T → A: 3-fold decrease in GSH affinity, 21-fold decrease in spermidine activity, and 17-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi538R → A: 6-fold decrease in GSH affinity, 2.4-fold decrease in spermidine activity, and 4-fold decrease in synthetase activity. 1 Publication1
    Mutagenesisi598R → A: 10-fold increase in GSH affinity, 9-fold decrease in spermidine activity, and 15-fold decrease in synthetase activity. 1 Publication1

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Initiator methionineiRemoved1 Publication
    ChainiPRO_00000704432 – 619Bifunctional glutathionylspermidine synthetase/amidaseAdd BLAST618

    Amino acid modifications

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Modified residuei59Cysteine sulfenic acid (-SOH); transient1 Publication1

    Post-translational modificationi

    Oxidation of Cys-59 to sulfenic acid during oxidative stress selectively inhibits the amidase activity which leads to a rapid increase in the amounts of intracellular Gsp and Gsp S-thiolated proteins (GspSSPs).1 Publication

    Keywords - PTMi

    Oxidation

    Proteomic databases

    PaxDbiP0AES0.
    PRIDEiP0AES0.

    Expressioni

    Inductioni

    Expression level is unaffected by H2O2; however Gsp rapidly accumulates in E.coli in the presence of H2O2.

    Interactioni

    Subunit structurei

    Homodimer.2 Publications

    Binary interactionsi

    WithEntry#Exp.IntActNotes
    fixBP315742EBI-557080,EBI-554030
    rhoP0AG304EBI-557080,EBI-545468

    Protein-protein interaction databases

    BioGridi4261180. 14 interactors.
    DIPiDIP-36018N.
    IntActiP0AES0. 14 interactors.
    MINTiMINT-1234290.
    STRINGi511145.b2988.

    Structurei

    Secondary structure

    1619
    Legend: HelixTurnBeta strandPDB Structure known for this area
    Show more details
    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Beta strandi15 – 19Combined sources5
    Turni20 – 22Combined sources3
    Beta strandi23 – 26Combined sources4
    Helixi35 – 40Combined sources6
    Helixi42 – 44Combined sources3
    Beta strandi45 – 48Combined sources4
    Beta strandi51 – 55Combined sources5
    Helixi59 – 71Combined sources13
    Beta strandi72 – 74Combined sources3
    Helixi81 – 86Combined sources6
    Beta strandi89 – 92Combined sources4
    Turni93 – 95Combined sources3
    Beta strandi98 – 100Combined sources3
    Beta strandi102 – 105Combined sources4
    Beta strandi108 – 110Combined sources3
    Beta strandi117 – 120Combined sources4
    Helixi124 – 126Combined sources3
    Turni127 – 129Combined sources3
    Beta strandi131 – 138Combined sources8
    Beta strandi140 – 146Combined sources7
    Beta strandi148 – 150Combined sources3
    Beta strandi162 – 170Combined sources9
    Beta strandi173 – 177Combined sources5
    Beta strandi179 – 182Combined sources4
    Beta strandi185 – 192Combined sources8
    Helixi206 – 209Combined sources4
    Beta strandi212 – 215Combined sources4
    Beta strandi229 – 231Combined sources3
    Helixi232 – 241Combined sources10
    Beta strandi245 – 247Combined sources3
    Beta strandi251 – 256Combined sources6
    Helixi257 – 283Combined sources27
    Helixi285 – 288Combined sources4
    Helixi289 – 291Combined sources3
    Helixi295 – 297Combined sources3
    Helixi298 – 307Combined sources10
    Helixi309 – 311Combined sources3
    Beta strandi314 – 322Combined sources9
    Beta strandi325 – 332Combined sources8
    Helixi339 – 343Combined sources5
    Helixi345 – 353Combined sources9
    Turni361 – 364Combined sources4
    Helixi365 – 374Combined sources10
    Beta strandi380 – 386Combined sources7
    Helixi390 – 405Combined sources16
    Beta strandi409 – 416Combined sources8
    Beta strandi423 – 425Combined sources3
    Beta strandi437 – 442Combined sources6
    Helixi444 – 453Combined sources10
    Turni456 – 458Combined sources3
    Beta strandi459 – 461Combined sources3
    Helixi475 – 479Combined sources5
    Beta strandi485 – 488Combined sources4
    Helixi490 – 493Combined sources4
    Turni494 – 496Combined sources3
    Helixi500 – 507Combined sources8
    Beta strandi517 – 520Combined sources4
    Helixi523 – 528Combined sources6
    Beta strandi530 – 534Combined sources5
    Turni539 – 542Combined sources4
    Beta strandi544 – 546Combined sources3
    Beta strandi552 – 555Combined sources4
    Turni559 – 562Combined sources4
    Beta strandi565 – 569Combined sources5
    Beta strandi579 – 588Combined sources10
    Beta strandi591 – 604Combined sources14
    Beta strandi609 – 612Combined sources4
    Beta strandi614 – 617Combined sources4

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    PDB entryMethodResolution (Å)ChainPositionsPDBsum
    2IO7X-ray2.70A/B1-619[»]
    2IO8X-ray2.10A/B1-619[»]
    2IO9X-ray2.20A/B1-619[»]
    2IOAX-ray2.80A/B1-619[»]
    2IOBX-ray2.20A/B1-619[»]
    3A2YX-ray1.95A1-197[»]
    3A2ZX-ray1.50A1-197[»]
    3A30X-ray2.20A1-197[»]
    3O98X-ray2.80A/B1-619[»]
    ProteinModelPortaliP0AES0.
    SMRiP0AES0.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiP0AES0.

    Family & Domainsi

    Domains and Repeats

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Domaini34 – 176Peptidase C51PROSITE-ProRule annotationAdd BLAST143

    Region

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Regioni2 – 195Gsp amidaseAdd BLAST194
    Regioni78 – 81Gsp binding4
    Regioni196 – 205Linker10
    Regioni206 – 619Gsp synthetaseAdd BLAST414

    Domaini

    The two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). The two domains expressed independently are folded and functional; liberation of the amidase domain from the synthetase domain highly activates the amidase activity.2 Publications

    Sequence similaritiesi

    In the C-terminal section; belongs to the glutathionylspermidine synthase preATP-grasp family.Curated
    Contains 1 peptidase C51 domain.PROSITE-ProRule annotation

    Phylogenomic databases

    eggNOGiENOG4106032. Bacteria.
    COG0754. LUCA.
    HOGENOMiHOG000124980.
    InParanoidiP0AES0.
    KOiK01460.
    OMAiPNHRYLL.

    Family and domain databases

    InterProiIPR007921. CHAP_dom.
    IPR005494. GSPS_pre-ATP-grasp-like_dom.
    IPR016185. PreATP-grasp_dom.
    [Graphical view]
    PfamiPF05257. CHAP. 1 hit.
    PF03738. GSP_synth. 1 hit.
    [Graphical view]
    SUPFAMiSSF52440. SSF52440. 1 hit.
    PROSITEiPS50911. CHAP. 1 hit.
    [Graphical view]

    Sequencei

    Sequence statusi: Complete.

    Sequence processingi: The displayed sequence is further processed into a mature form.

    P0AES0-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MSKGTTSQDA PFGTLLGYAP GGVAIYSSDY SSLDPQEYED DAVFRSYIDD
    60 70 80 90 100
    EYMGHKWQCV EFARRFLFLN YGVVFTDVGM AWEIFSLRFL REVVNDNILP
    110 120 130 140 150
    LQAFPNGSPR APVAGALLIW DKGGEFKDTG HVAIITQLHG NKVRIAEQNV
    160 170 180 190 200
    IHSPLPQGQQ WTRELEMVVE NGCYTLKDTF DDTTILGWMI QTEDTEYSLP
    210 220 230 240 250
    QPEIAGELLK ISGARLENKG QFDGKWLDEK DPLQNAYVQA NGQVINQDPY
    260 270 280 290 300
    HYYTITESAE QELIKATNEL HLMYLHATDK VLKDDNLLAL FDIPKILWPR
    310 320 330 340 350
    LRLSWQRRRH HMITGRMDFC MDERGLKVYE YNADSASCHT EAGLILERWA
    360 370 380 390 400
    EQGYKGNGFN PAEGLINELA GAWKHSRARP FVHIMQDKDI EENYHAQFME
    410 420 430 440 450
    QALHQAGFET RILRGLDELG WDAAGQLIDG EGRLVNCVWK TWAWETAFDQ
    460 470 480 490 500
    IREVSDREFA AVPIRTGHPQ NEVRLIDVLL RPEVLVFEPL WTVIPGNKAI
    510 520 530 540 550
    LPILWSLFPH HRYLLDTDFT VNDELVKTGY AVKPIAGRCG SNIDLVSHHE
    560 570 580 590 600
    EVLDKTSGKF AEQKNIYQQL WCLPKVDGKY IQVCTFTVGG NYGGTCLRGD
    610
    ESLVIKKESD IEPLIVVKK
    Length:619
    Mass (Da):70,532
    Last modified:December 20, 2005 - v1
    Checksum:i07FB43D8A0B2933C
    GO

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    U23148 Genomic DNA. Translation: AAC43339.1.
    U28377 Genomic DNA. Translation: AAA69155.1.
    U00096 Genomic DNA. Translation: AAC76024.1.
    AP009048 Genomic DNA. Translation: BAE77049.1.
    PIRiA57538.
    RefSeqiNP_417462.1. NC_000913.3.
    WP_001297309.1. NZ_LN832404.1.

    Genome annotation databases

    EnsemblBacteriaiAAC76024; AAC76024; b2988.
    BAE77049; BAE77049; BAE77049.
    GeneIDi947474.
    KEGGiecj:JW2956.
    eco:b2988.
    PATRICi32121392. VBIEscCol129921_3083.

    Cross-referencesi

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    U23148 Genomic DNA. Translation: AAC43339.1.
    U28377 Genomic DNA. Translation: AAA69155.1.
    U00096 Genomic DNA. Translation: AAC76024.1.
    AP009048 Genomic DNA. Translation: BAE77049.1.
    PIRiA57538.
    RefSeqiNP_417462.1. NC_000913.3.
    WP_001297309.1. NZ_LN832404.1.

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    PDB entryMethodResolution (Å)ChainPositionsPDBsum
    2IO7X-ray2.70A/B1-619[»]
    2IO8X-ray2.10A/B1-619[»]
    2IO9X-ray2.20A/B1-619[»]
    2IOAX-ray2.80A/B1-619[»]
    2IOBX-ray2.20A/B1-619[»]
    3A2YX-ray1.95A1-197[»]
    3A2ZX-ray1.50A1-197[»]
    3A30X-ray2.20A1-197[»]
    3O98X-ray2.80A/B1-619[»]
    ProteinModelPortaliP0AES0.
    SMRiP0AES0.
    ModBaseiSearch...
    MobiDBiSearch...

    Protein-protein interaction databases

    BioGridi4261180. 14 interactors.
    DIPiDIP-36018N.
    IntActiP0AES0. 14 interactors.
    MINTiMINT-1234290.
    STRINGi511145.b2988.

    Protein family/group databases

    MEROPSiC51.A01.

    Proteomic databases

    PaxDbiP0AES0.
    PRIDEiP0AES0.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Genome annotation databases

    EnsemblBacteriaiAAC76024; AAC76024; b2988.
    BAE77049; BAE77049; BAE77049.
    GeneIDi947474.
    KEGGiecj:JW2956.
    eco:b2988.
    PATRICi32121392. VBIEscCol129921_3083.

    Organism-specific databases

    EchoBASEiEB2720.
    EcoGeneiEG12882. gss.

    Phylogenomic databases

    eggNOGiENOG4106032. Bacteria.
    COG0754. LUCA.
    HOGENOMiHOG000124980.
    InParanoidiP0AES0.
    KOiK01460.
    OMAiPNHRYLL.

    Enzyme and pathway databases

    UniPathwayiUPA00204.
    UPA00819.
    BioCyciEcoCyc:GSP-MONOMER.
    ECOL316407:JW2956-MONOMER.
    MetaCyc:GSP-MONOMER.
    BRENDAi3.5.1.78. 2026.
    6.3.1.8. 2026.

    Miscellaneous databases

    EvolutionaryTraceiP0AES0.
    PROiP0AES0.

    Family and domain databases

    InterProiIPR007921. CHAP_dom.
    IPR005494. GSPS_pre-ATP-grasp-like_dom.
    IPR016185. PreATP-grasp_dom.
    [Graphical view]
    PfamiPF05257. CHAP. 1 hit.
    PF03738. GSP_synth. 1 hit.
    [Graphical view]
    SUPFAMiSSF52440. SSF52440. 1 hit.
    PROSITEiPS50911. CHAP. 1 hit.
    [Graphical view]
    ProtoNetiSearch...

    Entry informationi

    Entry nameiGSP_ECOLI
    AccessioniPrimary (citable) accession number: P0AES0
    Secondary accession number(s): P43675, Q2M9K7
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: December 20, 2005
    Last sequence update: December 20, 2005
    Last modified: November 2, 2016
    This is version 93 of the entry and version 1 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Miscellaneous

    Gsp forms mixed disulfides with the thiols of a variety of E.coli proteins. These mixed disulfides represent a previously uncharacterized type of post-translational modification. The level of these proteins is increased by oxidative stress, which implies that Gsp might protect protein thiols against irreversible oxidation (PubMed:20530482).1 Publication
    No metal ion is required for the amidase activity.1 Publication
    Gsp hydrolysis to GSH and spermidine proceeds with formation of a glutathionyl acyl-enzyme intermediate, utilizing a cysteine residue as the catalytic nucleophile (PubMed:9398217). For Gsp synthesis, GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine (PubMed:17124497).2 Publications

    Keywords - Technical termi

    3D-structure, Complete proteome, Direct protein sequencing, Multifunctional enzyme, Reference proteome

    Documents

    1. Escherichia coli
      Escherichia coli (strain K12): entries and cross-references to EcoGene
    2. PATHWAY comments
      Index of metabolic and biosynthesis pathways
    3. PDB cross-references
      Index of Protein Data Bank (PDB) cross-references
    4. Peptidase families
      Classification of peptidase families and list of entries
    5. SIMILARITY comments
      Index of protein domains and families

    Similar proteinsi

    Links to similar proteins from the UniProt Reference Clusters (UniRef) at 100%, 90% and 50% sequence identity:
    100%UniRef100 combines identical sequences and sub-fragments with 11 or more residues from any organism into one UniRef entry.
    90%UniRef90 is built by clustering UniRef100 sequences that have at least 90% sequence identity to, and 80% overlap with, the longest sequence (a.k.a seed sequence).
    50%UniRef50 is built by clustering UniRef90 seed sequences that have at least 50% sequence identity to, and 80% overlap with, the longest sequence in the cluster.