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MetaCyc Enzyme: glutathione reductase

Gene: gor Accession Numbers: EG10412 (MetaCyc), b3500, ECK3485

Synonyms: gorA

Species: Escherichia coli K-12 substr. MG1655

Subunit composition of glutathione reductase = [Gor]2
         glutathione reductase (NADPH) = Gor

Summary:
Glutathione reductase (Gor) is a flavoprotein that reduces oxidized glutathione to form reduced glutathione (GSH), the major nonprotein sulfhydryl in living organisms. Glutathione takes part in many different intracellular processes, including maintenance of reduced thiol groups, protection from oxygen-induced cell damage, and generation of deoxyribonucleotide precursors for DNA synthesis. Glutathione reductase together with NADPH, glutathione, and glutaredoxin make up a second hydrogen donor system in E. coli in addition to the thioredoxin system [Holmgren76] (see glutathione redox reactions II and thioredoxin pathway).

Glutathione reductase is a member of the family of flavin-dependent pyridine nucleotide-disulfide oxidoreductases. Other members of this family include thioredoxin reductase, lipoamide dehydrogenase, 2-oxopropyl-CoM reductase (carboxylating), trypanothione reductase, and mercury(II) reductase [Mittl94].

In mutants lacking glutathione reductase activity the ratio of reduced to oxidized glutathione is not significantly changed suggesting that either alternative enzymes exist, or the thioredoxin pathway may also participate [Tuggle85, Ritz01a]. These mutations also cause no growth defect, although the activity of glutathione reductase appears to be essential to maintaining a high glutathione content [Davis82, Kunert90].

The native E. coli enzyme has been purified, characterized, and its redox interconversions studied [Pigiet77, Mata84, Mata85, Mata85a, Arscott89]. The gene was cloned and the recombinant enzyme was also studied [Greer86, Scrutton87, Perham87a]. A number of site-directed mutagenesis studies have investigated the structure and catalytic mechanism of Gor [Scrutton88, Deonarain89, Deonarain90, Scrutton90, Deonarain92, Deonarain92a, Rietveld94, vandenBerg98]. The E. coli enzyme has also been subjected to comparative kinetic analysis with eukaryotic enzymes [Vanoni90, Veine98]. Cell-free synthesis of active E. coli Gor has also been demonstrated [Knapp04].

Crystal structures of the wild-type and mutant Gor enzymes have been determined [Ermler91, Mittl93, Mittl94a, Mittl94]. The enzyme was shown to catalyze the reduction of glutathione disulfide via two half-reactions. In the reductive half-reaction FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox-active disulfide. The oxidative half-reaction involves dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide [Rietveld94]. More recently, it was shown that E. coli Gor can catalyze the NADPH-dependent reduction of AuCl4- to form gold nanoparticles at the active site that were tightly bound through the catalytic cysteines, and could be removed from the active site by thiol reagents [Scott08].

Review: [Ritz01a]
In a gene expression analysis, RpoS positively regulated gor gene in the stationary phase. Its effect could be indirect [BeckerHapak95].

Locations: membrane, cytosol

Map Position: [3,644,322 -> 3,645,674]

Molecular Weight of Polypeptide: 48.772 kD (from nucleotide sequence), 55.0 kD (experimental) [Mata84 ]

Molecular Weight of Multimer: 109.0 kD (experimental) [Mata84]

pI: 5.97

Unification Links: ASAP:ABE-0011432 , CGSC:665 , EchoBASE:EB0407 , EcoGene:EG10412 , EcoliWiki:b3500 , ModBase:P06715 , OU-Microarray:b3500 , PortEco:gor , PR:PRO_000022829 , Pride:P06715 , Protein Model Portal:P06715 , RefSeq:NP_417957 , RegulonDB:EG10412 , SMR:P06715 , String:511145.b3500 , UniProt:P06715

Relationship Links: InterPro:IN-FAMILY:IPR001327 , InterPro:IN-FAMILY:IPR004099 , InterPro:IN-FAMILY:IPR006322 , InterPro:IN-FAMILY:IPR012999 , InterPro:IN-FAMILY:IPR013027 , InterPro:IN-FAMILY:IPR016156 , InterPro:IN-FAMILY:IPR023753 , PDB:Structure:1GER , PDB:Structure:1GES , PDB:Structure:1GET , PDB:Structure:1GEU , Pfam:IN-FAMILY:PF00070 , Pfam:IN-FAMILY:PF02852 , Pfam:IN-FAMILY:PF07992 , Prints:IN-FAMILY:PR00368 , Prosite:IN-FAMILY:PS00076

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0006749 - glutathione metabolic process Inferred by computational analysis [GOA01]
GO:0045454 - cell redox homeostasis Inferred by computational analysis [GOA01]
GO:0055114 - oxidation-reduction process Inferred by computational analysis [UniProtGOA11, GOA01]
Molecular Function: GO:0004362 - glutathione-disulfide reductase activity Inferred from experiment Inferred by computational analysis [GOA01a, GOA01, Mata84]
GO:0042803 - protein homodimerization activity Inferred from experiment [Mata84]
GO:0071949 - FAD binding Inferred from experiment [Pigiet77]
GO:0016491 - oxidoreductase activity Inferred by computational analysis [UniProtGOA11, GOA01]
GO:0016668 - oxidoreductase activity, acting on a sulfur group of donors, NAD(P) as acceptor Inferred by computational analysis [GOA01]
GO:0050660 - flavin adenine dinucleotide binding Inferred by computational analysis [GOA01]
GO:0050661 - NADP binding Inferred by computational analysis [GOA01]
Cellular Component: GO:0005829 - cytosol Inferred from experiment [Pigiet77]
GO:0016020 - membrane Inferred from experiment [Lasserre06]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11a, UniProtGOA11]

Gene Class: UNCLASSIFIED

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


Enzymatic reaction of: glutathione reductase

Synonyms: GR, GRase, NADPH:oxidized-glutathione oxidoreductase

EC Number: 1.8.1.7

glutathione disulfide + NADPH + H+ <=> 2 glutathione + NADP+

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.

In Pathways: glutathione redox reactions II

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

Summary:
The Km values for NADPH, NADH, and glutathione disulfide at pH 7.5 were 16 μM, 377 μM and 66 μM, respectively. The optimum pH was 7.5 with NADPH and 4.5 with NADH as reductant [Mata84].

Cofactors or Prosthetic Groups: FAD [Mata84]

Inhibitors (Unknown Mechanism): p-hydroxymercuribenzoate [Mata84, Helmward89] , NADPH [Mata84, Comment 1] , glutathione disulfide [Mata84, Comment 2]

Kinetic Parameters:

Substrate
Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
Citations
NADPH
25.0
[Scrutton87, BRENDA14]
NADPH
16.0
[Mata84, BRENDA14]
glutathione disulfide
70.0
[Scrutton87, BRENDA14]
glutathione disulfide
66.0
[Mata84, BRENDA14]
glutathione disulfide
61.0
733.3
[Henderson91, BRENDA14]

T(opt): 37 °C [BRENDA14, Knapp04]

pH(opt) (forward direction): 7.5 [Mata84]

pH(opt): 4.5 [BRENDA14, Mata84]


Sequence Features

Feature Class Location Citations Comment
Nucleotide-Phosphate-Binding-Region 34 -> 41
[UniProt10a]
UniProt: FAD;
Disulfide-Bond-Site 47, 42
[Mittl94, UniProt11]
UniProt: Redox-active.
Active-Site 439
[UniProt10a]
UniProt: Proton acceptor;

History:
10/20/97 Gene b3500 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10412; confirmed by SwissProt match.


References

Arscott89: Arscott LD, Drake DM, Williams CH (1989). "Inactivation-reactivation of two-electron reduced Escherichia coli glutathione reductase involving a dimer-monomer equilibrium." Biochemistry 28(8);3591-8. PMID: 2663073

BeckerHapak95: Becker-Hapak M, Eisenstark A (1995). "Role of rpoS in the regulation of glutathione oxidoreductase (gor) in Escherichia coli." FEMS Microbiol Lett 134(1);39-44. PMID: 8593953

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

Davis82: Davis NK, Greer S, Jones-Mortimer MC, Perham RN (1982). "Isolation and mapping of glutathione reductase-negative mutants of Escherichia coli K12." J Gen Microbiol 128(7);1631-4. PMID: 6214613

Deonarain89: Deonarain MP, Berry A, Scrutton NS, Perham RN (1989). "Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99." Biochemistry 28(25);9602-7. PMID: 2558727

Deonarain90: Deonarain MP, Scrutton NS, Berry A, Perham RN (1990). "Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli." Proc R Soc Lond B Biol Sci 1990;241(1302);179-86. PMID: 1979442

Deonarain92: Deonarain MP, Scrutton NS, Perham RN (1992). "Engineering surface charge. 1. A method for detecting subunit exchange in Escherichia coli glutathione reductase." Biochemistry 31(5);1491-7. PMID: 1737008

Deonarain92a: Deonarain MP, Scrutton NS, Perham RN (1992). "Engineering surface charge. 2. A method for purifying heterodimers of Escherichia coli glutathione reductase." Biochemistry 31(5);1498-504. PMID: 1737009

Ermler91: Ermler U, Schulz GE (1991). "The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution." Proteins 9(3);174-9. PMID: 2006135

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.

Greer86: Greer S, Perham RN (1986). "Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases." Biochemistry 25(9);2736-42. PMID: 3521741

Helmward89: Helmward Z "Handbook of Enzyme Inhibitors. 2nd, revised and enlarged edition." Weinheim, Federal Republic of Germany ; New York, NY, USA , 1989.

Henderson91: Henderson GB, Murgolo NJ, Kuriyan J, Osapay K, Kominos D, Berry A, Scrutton NS, Hinchliffe NW, Perham RN, Cerami A (1991). "Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction." Proc Natl Acad Sci U S A 88(19);8769-73. PMID: 1924337

Holmgren76: Holmgren A (1976). "Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione." Proc Natl Acad Sci U S A 1976;73(7);2275-9. PMID: 7783

Knapp04: Knapp KG, Swartz JR (2004). "Cell-free production of active E. coli thioredoxin reductase and glutathione reductase." FEBS Lett 559(1-3);66-70. PMID: 14960309

Kunert90: Kunert KJ, Cresswell CF, Schmidt A, Mullineaux PM, Foyer CH (1990). "Variations in the activity of glutathione reductase and the cellular glutathione content in relation to sensitivity to methylviologen in Escherichia coli." Arch Biochem Biophys 1990;282(2);233-8. PMID: 2241146

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

Mata84: Mata AM, Pinto MC, Lopez-Barea J (1984). "Purification by affinity chromatography of glutathione reductase (EC 1.6.4.2) from Escherichia coli and characterization of such enzyme." Z Naturforsch [C] 39(9-10);908-15. PMID: 6393625

Mata85: Mata AM, Pinto MC, Lopez-Barea J (1985). "Redox interconversion of glutathione reductase from Escherichia coli. A study with pure enzyme and cell-free extracts." Mol Cell Biochem 67(1);65-76. PMID: 3894932

Mata85a: Mata AM, Pinto MC, Lopez-Barea J (1985). "Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells." Mol Cell Biochem 68(2);121-30. PMID: 3908906

Mittl93: Mittl PR, Berry A, Scrutton NS, Perham RN, Schulz GE (1993). "Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant." J Mol Biol 231(2);191-5. PMID: 8510142

Mittl94: Mittl PR, Schulz GE (1994). "Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes." Protein Sci 3(5);799-809. PMID: 8061609

Mittl94a: Mittl PR, Berry A, Scrutton NS, Perham RN, Schulz GE (1994). "Anatomy of an engineered NAD-binding site." Protein Sci 3(9);1504-14. PMID: 7833810

Perham87a: Perham RN (1987). "Glutathione reductase from Escherichia coli: mutation, cloning and sequence analysis of the gene." Biochem Soc Trans 15(4);730-3. PMID: 3315774

Pigiet77: Pigiet VP, Conley RR (1977). "Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography." J Biol Chem 252(18);6367-72. PMID: 330529

Rietveld94: Rietveld P, Arscott LD, Berry A, Scrutton NS, Deonarain MP, Perham RN, Williams CH (1994). "Reductive and oxidative half-reactions of glutathione reductase from Escherichia coli." Biochemistry 33(46);13888-95. PMID: 7947797

Ritz01a: Ritz D, Beckwith J (2001). "Roles of thiol-redox pathways in bacteria." Annu Rev Microbiol 55;21-48. PMID: 11544348

Scott08: Scott D, Toney M, Muzikar M (2008). "Harnessing the mechanism of glutathione reductase for synthesis of active site bound metallic nanoparticles and electrical connection to electrodes." J Am Chem Soc 130(3);865-74. PMID: 18166048

Scrutton87: Scrutton NS, Berry A, Perham RN (1987). "Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli." Biochem J 245(3);875-80. PMID: 3311037

Scrutton88: Scrutton NS, Berry A, Perham RN (1988). "Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli." FEBS Lett 241(1-2);46-50. PMID: 3058515

Scrutton90: Scrutton NS, Berry A, Deonarain MP, Perham RN (1990). "Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo." Proc Biol Sci 242(1305);217-24. PMID: 1983037

Tuggle85: Tuggle CK, Fuchs JA (1985). "Glutathione reductase is not required for maintenance of reduced glutathione in Escherichia coli K-12." J Bacteriol 162(1);448-50. PMID: 3884598

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

UniProt11: 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."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

vandenBerg98: van den Berg PA, van Hoek A, Walentas CD, Perham RN, Visser AJ (1998). "Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase." Biophys J 74(4);2046-58. PMID: 9545063

Vanoni90: Vanoni MA, Wong KK, Ballou DP, Blanchard JS (1990). "Glutathione reductase: comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes." Biochemistry 29(24);5790-6. PMID: 2200516

Veine98: Veine DM, Arscott LD, Williams CH (1998). "Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis." Biochemistry 37(44);15575-82. PMID: 9799522


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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
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