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P0AES0 (GSP_ECOLI) Reviewed, UniProtKB/Swiss-Prot

Last modified July 9, 2014. Version 77. Feed History...

Clusters with 100%, 90%, 50% identity | Documents (5) | Third-party data text xml rdf/xml gff fasta
to top of pageNames·Attributes·General annotation·Ontologies·Interactions·Sequence annotation·Sequences·References·Cross-refs·Entry info·DocumentsCustomize order

Names and origin

Protein namesRecommended name:
Bifunctional glutathionylspermidine synthetase/amidase

Short name=GspSA

Including the following 2 domains:

  1. Glutathionylspermidine amidase
    Short name=Gsp amidase
    EC=3.5.1.78
    Alternative name(s):
    Glutathionylspermidine amidohydrolase [spermidine-forming]
  2. Glutathionylspermidine synthetase
    Short name=Gsp synthetase
    EC=6.3.1.8
    Alternative name(s):
    Glutathione:spermidine ligase [ADP-forming]
    Gsp synthase
Gene names
Name:gss
Synonyms:gsp
Ordered Locus Names:b2988, JW2956
OrganismEscherichia coli (strain K12) [Reference proteome] [HAMAP]
Taxonomic identifier83333 [NCBI]
Taxonomic lineageBacteriaProteobacteriaGammaproteobacteriaEnterobacterialesEnterobacteriaceaeEscherichia

Protein attributes

Sequence length619 AA.
Sequence statusComplete.
Sequence processingThe displayed sequence is further processed into a mature form.
Protein existenceEvidence at protein level

General annotation (Comments)

Function

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). Ref.1 Ref.5 Ref.6 Ref.8

Catalytic activity

Glutathione + spermidine + ATP = glutathionylspermidine + ADP + phosphate. Ref.1 Ref.5 Ref.6 Ref.8

Glutathionylspermidine + H2O = glutathione + spermidine. Ref.1 Ref.5 Ref.6 Ref.8

Enzyme regulation

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. Ref.4 Ref.5 Ref.8

Pathway

Sulfur metabolism; glutathione metabolism.

Amine and polyamine metabolism; spermidine metabolism.

Subunit structure

Homodimer. Ref.1 Ref.7

Induction

Expression level is unaffected by H2O2; however Gsp rapidly accumulates in E.coli in the presence of H2O2. Ref.4 Ref.5 Ref.8

Domain

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. Ref.5 Ref.7

Post-translational modification

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).

Disruption phenotype

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. Ref.6 Ref.8

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 (Ref.8).

No metal ion is required for the amidase activity (Ref.5).

Gsp hydrolysis to GSH and spermidine proceeds with formation of a glutathionyl acyl-enzyme intermediate, utilizing a cysteine residue as the catalytic nucleophile (Ref.4). 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 (Ref.7).

Sequence similarities

In the C-terminal section; belongs to the glutathionylspermidine synthase preATP-grasp family.

Contains 1 peptidase C51 domain.

Biophysicochemical properties

Kinetic parameters:

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 (Ref.1).

KM=100 µM for ATP (at pH 6.8) (Ref.1) Ref.1 Ref.7

KM=800 µM for glutathione (at pH 6.8) (Ref.1)

KM=218 µM for glutathione (Ref.7)

KM=60 µM for spermidine (at pH 6.8) (Ref.1)

KM=20 µM for spermidine (at pH 7.5) (Ref.1)

KM=76 µM for spermidine (Ref.7)

KM=900 µM for glutathionylspermidine (at pH 7.5) (Ref.1)

pH dependence:

Optimum pH is around 6.8 for Gsp synthetase activity (Ref.1).

Binary interactions

Sequence annotation (Features)

Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifier

Molecule processing

Initiator methionine11Removed Ref.1
Chain2 – 619618Bifunctional glutathionylspermidine synthetase/amidase
PRO_0000070443

Regions

Domain34 – 176143Peptidase C51
Nucleotide binding316 – 3183ATP
Nucleotide binding539 – 5402ATP
Nucleotide binding568 – 5714ATP
Nucleotide binding603 – 6053ATP
Region2 – 195194Gsp amidase
Region78 – 814Gsp binding
Region196 – 20510Linker
Region206 – 619414Gsp synthetase

Sites

Active site591S-(gamma-glutamyl-cysteinyl-glycyl)-cysteine intermediate Ref.4 Ref.9
Metal binding3181Magnesium 1
Metal binding3301Magnesium 1
Metal binding3301Magnesium 2
Metal binding3321Magnesium 2
Binding site581Gsp
Binding site641Gsp
Binding site1491Gsp
Binding site3161Glutathione
Binding site3351Glutathione
Binding site3911Spermidine
Binding site3921Glutathione
Binding site4461Glutathione
Binding site4981ATP
Binding site5331ATP
Binding site5821ATP
Binding site6101Spermidine
Site1311Increases nucleophilicity of active site Cys; for amidase activity
Site3161Transition state stabilizer; for synthetase activity

Amino acid modifications

Modified residue591Cysteine sulfenic acid (-SOH); transient

Experimental info

Mutagenesis591C → A: Loss of amidase activity. Ref.4
Mutagenesis1731C → A: No effect on amidase activity. Ref.4
Mutagenesis3161R → E: Loss of synthetase activity.
Mutagenesis3351S → A: 3.6-fold decrease in GSH affinity, 1.6-fold decrease in spermidine activity, and 1.3-fold decrease in synthetase activity. Ref.7
Mutagenesis3371S → A: No effect on GSH and spermidine affinity, but 2-fold decrease in synthetase activity. Ref.7
Mutagenesis3381C → A: 10-fold decrease in GSH affinity, 5-fold decrease in spermidine activity, but no effect on synthetase activity. Ref.7
Mutagenesis3911E → A: 2-fold decrease in GSH affinity, 60-fold decrease in spermidine activity, and 10-fold decrease in synthetase activity. Ref.7
Mutagenesis3921E → A: 33-fold decrease in GSH affinity, 13-fold decrease in spermidine activity, and 6-fold decrease in synthetase activity. Ref.7
Mutagenesis4411T → A: 3-fold decrease in GSH affinity, 21-fold decrease in spermidine activity, and 17-fold decrease in synthetase activity. Ref.7
Mutagenesis5381R → A: 6-fold decrease in GSH affinity, 2.4-fold decrease in spermidine activity, and 4-fold decrease in synthetase activity. Ref.7
Mutagenesis5981R → A: 10-fold increase in GSH affinity, 9-fold decrease in spermidine activity, and 15-fold decrease in synthetase activity. Ref.7

Secondary structure

............................................................................................................................ 619
Helix Strand Turn

Details...

Sequences

Sequence LengthMass (Da)Tools
P0AES0 [UniParc].

Last modified December 20, 2005. Version 1.
Checksum: 07FB43D8A0B2933C

FASTA61970,532
        10         20         30         40         50         60 
MSKGTTSQDA PFGTLLGYAP GGVAIYSSDY SSLDPQEYED DAVFRSYIDD EYMGHKWQCV 

        70         80         90        100        110        120 
EFARRFLFLN YGVVFTDVGM AWEIFSLRFL REVVNDNILP LQAFPNGSPR APVAGALLIW 

       130        140        150        160        170        180 
DKGGEFKDTG HVAIITQLHG NKVRIAEQNV IHSPLPQGQQ WTRELEMVVE NGCYTLKDTF 

       190        200        210        220        230        240 
DDTTILGWMI QTEDTEYSLP QPEIAGELLK ISGARLENKG QFDGKWLDEK DPLQNAYVQA 

       250        260        270        280        290        300 
NGQVINQDPY HYYTITESAE QELIKATNEL HLMYLHATDK VLKDDNLLAL FDIPKILWPR 

       310        320        330        340        350        360 
LRLSWQRRRH HMITGRMDFC MDERGLKVYE YNADSASCHT EAGLILERWA EQGYKGNGFN 

       370        380        390        400        410        420 
PAEGLINELA GAWKHSRARP FVHIMQDKDI EENYHAQFME QALHQAGFET RILRGLDELG 

       430        440        450        460        470        480 
WDAAGQLIDG EGRLVNCVWK TWAWETAFDQ IREVSDREFA AVPIRTGHPQ NEVRLIDVLL 

       490        500        510        520        530        540 
RPEVLVFEPL WTVIPGNKAI LPILWSLFPH HRYLLDTDFT VNDELVKTGY AVKPIAGRCG 

       550        560        570        580        590        600 
SNIDLVSHHE EVLDKTSGKF AEQKNIYQQL WCLPKVDGKY IQVCTFTVGG NYGGTCLRGD 

       610 
ESLVIKKESD IEPLIVVKK 

« Hide

References

« Hide 'large scale' references
[1]"Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase."
Bollinger J.M. Jr., Kwon D.S., Huisman G.W., Kolter R., Walsh C.T.
J. Biol. Chem. 270:14031-14041(1995) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-7, FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT.
Strain: B and K12.
[2]"The complete genome sequence of Escherichia coli K-12."
Blattner F.R., Plunkett G. III, Bloch C.A., Perna N.T., Burland V., Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F., Gregor J., Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., Mau B., Shao Y.
Science 277:1453-1462(1997) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
Strain: K12 / MG1655 / ATCC 47076.
[3]"Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110."
Hayashi K., Morooka N., Yamamoto Y., Fujita K., Isono K., Choi S., Ohtsubo E., Baba T., Wanner B.L., Mori H., Horiuchi T.
Mol. Syst. Biol. 2:E1-E5(2006) [PubMed] [Europe PMC] [Abstract]
Cited for: NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
Strain: K12 / W3110 / ATCC 27325 / DSM 5911.
[4]"Evidence for a glutathionyl-enzyme intermediate in the amidase activity of the bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli."
Lin C.H., Kwon D.S., Bollinger J.M. Jr., Walsh C.T.
Biochemistry 36:14930-14938(1997) [PubMed] [Europe PMC] [Abstract]
Cited for: REACTION MECHANISM OF AMIDASE ACTIVITY, ACTIVE SITE AT CYS-59, ENZYME REGULATION, MUTAGENESIS OF CYS-59 AND CYS-173.
[5]"Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains."
Kwon D.S., Lin C.H., Chen S., Coward J.K., Walsh C.T., Bollinger J.M. Jr.
J. Biol. Chem. 272:2429-2436(1997) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, CATALYTIC ACTIVITY, DOMAIN, SUBSTRATE SPECIFICITY, ENZYME REGULATION.
[6]"Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC."
Sui L., Warren J.C., Russell J.P., Stourman N.V.
Int. J. Biochem. Mol. Biol. 3:302-312(2012) [PubMed] [Europe PMC] [Abstract]
Cited for: FUNCTION, CATALYTIC ACTIVITY, DISRUPTION PHENOTYPE.
Strain: K12.
[7]"Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase."
Pai C.H., Chiang B.Y., Ko T.P., Chou C.C., Chong C.M., Yen F.J., Chen S., Coward J.K., Wang A.H., Lin C.H.
EMBO J. 25:5970-5982(2006) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) OF APOENZYME AND IN COMPLEXES WITH SUBSTRATE; PRODUCT AND INHIBITOR, DOMAIN BOUNDARIES, SUBUNIT, REACTION MECHANISM OF SYNTHETASE ACTIVITY, KINETIC PARAMETERS, SITE AT ARG-316, MUTAGENESIS OF SER-335; SER-337; CYS-338; GLU-391; GLU-392; THR-441; ARG-538 AND ARG-598.
[8]"Protein S-thiolation by glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetase/amidase in redox regulation."
Chiang B.Y., Chen T.C., Pai C.H., Chou C.C., Chen H.H., Ko T.P., Hsu W.H., Chang C.Y., Wu W.F., Wang A.H., Lin C.H.
J. Biol. Chem. 285:25345-25353(2010) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (1.50 ANGSTROMS) OF 1-197, FUNCTION, ROLE IN REDOX REGULATION, CATALYTIC ACTIVITY, ENZYME REGULATION, OXIDATION AT CYS-59, DISRUPTION PHENOTYPE.
[9]"Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases."
Pai C.H., Wu H.J., Lin C.H., Wang A.H.
Protein Sci. 20:557-566(2011) [PubMed] [Europe PMC] [Abstract]
Cited for: X-RAY CRYSTALLOGRAPHY (1.95 ANGSTROMS) OF MUTANT ALA-59 IN COMPLEXES WITH ADP; GLUTATHIONYLSPERMIDINE AND MAGNESIUM, ACTIVE SITES, CATALYTIC MECHANISM OF AMIDASE ACTIVITY.
+Additional computationally mapped references.

Cross-references

Sequence databases

EMBL
GenBank
DDBJ
U23148 Genomic DNA. Translation: AAC43339.1.
U28377 Genomic DNA. Translation: AAA69155.1.
U00096 Genomic DNA. Translation: AAC76024.1.
AP009048 Genomic DNA. Translation: BAE77049.1.
PIRA57538.
RefSeqNP_417462.1. NC_000913.3.
YP_491185.1. NC_007779.1.

3D structure databases

PDBe
RCSB-PDB
PDBj
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[»]
ModBaseSearch...
MobiDBSearch...

Protein-protein interaction databases

DIPDIP-36018N.
IntActP0AES0. 14 interactions.
MINTMINT-1234290.
STRING511145.b2988.

Protein family/group databases

MEROPSC51.A01.

Proteomic databases

PaxDbP0AES0.
PRIDEP0AES0.

Protocols and materials databases

StructuralBiologyKnowledgebaseSearch...

Genome annotation databases

EnsemblBacteriaAAC76024; AAC76024; b2988.
BAE77049; BAE77049; BAE77049.
GeneID12932186.
947474.
KEGGecj:Y75_p2917.
eco:b2988.
PATRIC32121392. VBIEscCol129921_3083.

Organism-specific databases

EchoBASEEB2720.
EcoGeneEG12882. gss.

Phylogenomic databases

eggNOGCOG0754.
HOGENOMHOG000124980.
KOK01460.
OMAYMGYKWQ.
OrthoDBEOG66XB93.

Enzyme and pathway databases

BioCycEcoCyc:GSP-MONOMER.
ECOL316407:JW2956-MONOMER.
MetaCyc:GSP-MONOMER.
UniPathwayUPA00204.
UPA00819.

Gene expression databases

GenevestigatorP0AES0.

Family and domain databases

InterProIPR007921. CHAP_dom.
IPR005494. GSPS_pre-ATP-grasp-like_dom.
IPR016185. PreATP-grasp_dom.
[Graphical view]
PfamPF05257. CHAP. 1 hit.
PF03738. GSP_synth. 1 hit.
[Graphical view]
SUPFAMSSF52440. SSF52440. 1 hit.
PROSITEPS50911. CHAP. 1 hit.
[Graphical view]
ProtoNetSearch...

Other

EvolutionaryTraceP0AES0.
PROP0AES0.

Entry information

Entry nameGSP_ECOLI
AccessionPrimary (citable) accession number: P0AES0
Secondary accession number(s): P43675, Q2M9K7
Entry history
Integrated into UniProtKB/Swiss-Prot: December 20, 2005
Last sequence update: December 20, 2005
Last modified: July 9, 2014
This is version 77 of the entry and version 1 of the sequence. [Complete history]
Entry statusReviewed (UniProtKB/Swiss-Prot)
Annotation programProkaryotic Protein Annotation Program

Relevant documents

SIMILARITY comments

Index of protein domains and families

Peptidase families

Classification of peptidase families and list of entries

PDB cross-references

Index of Protein Data Bank (PDB) cross-references

PATHWAY comments

Index of metabolic and biosynthesis pathways

Escherichia coli

Escherichia coli (strain K12): entries and cross-references to EcoGene