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Literature summary extracted from

  • Hopkinson, R.J.; Leung, I.K.; Smart, T.J.; Rose, N.R.; Henry, L.; Claridge, T.D.; Schofield, C.J.
    Studies on the glutathione-dependent formaldehyde-activating enzyme from Paracoccus denitrificans (2015), PLoS ONE, 10, e0145085 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.4.1.22 gene gfa, recombinant expression of His10-tagged enzyme in Escherichia coli strain BL21(DE3), over expression of seleno-methionine-labeled enzyme Paracoccus denitrificans

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.4.1.22 analysis of seleno-methionine-labeled enzyme GFA X-ray crystal structures from Paracoccus denitrificans, PDB IDs 1X6M and 1XA8. Crystallization of purified recombinant detagged enzyme, free or as Gfa-glutathione complex, vapor diffusion by mixing 0.002 ml of 9 mg/ml protein in 20 mM HEPES, pH 7.0, and 100 mM NaCl with 0.002 ml of reservoir solution containing 2.0-2.1 M (NH4)2SO4, 4% PEG 400, and 0.1 M HEPES, pH 7.4, for the enzyme complex crystals, 10 mM of the reduced form of L-glutathione and 5 mM 2-mercaptoethanol are added to the reservoir solution, X-ray diffraction structure determination and analysis at 2.35 A resolution Paracoccus denitrificans

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.4.1.22 Zn2+ required, the GFA domain contains two zinc binding sites, one zinc ion is coordinated by four cysteinyl thiols (C31, C33, C99 and C102) in a tetrahedral geometry, whereas the other zinc ion is coordinated by three cysteinyl thiols (C52, C54 and C57) in a trigonal planar geometry. GSH binding induces translocation of the second zinc ion. Manual model building of the catalytic zinc site Paracoccus denitrificans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.4.1.22 5,6,7,8-tetrahydromethanopterin + formaldehyde Paracoccus denitrificans
-
 5,10-methylenetetrahydromethanopterin + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.4.1.22 Paracoccus denitrificans Q51669
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.4.1.22 recombinant His10-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, and gel filtration Paracoccus denitrificans

Reaction

EC Number Reaction Comment Organism Reaction ID
4.4.1.22 S-(hydroxymethyl)glutathione = glutathione + formaldehyde GSH reacts with HCHO via its nucleophilic thiol group to form the intermediate S-hydroxymethylglutathione (HMG). The catalysis of enzyme glutathione-dependent formaldehyde-activating enzyme (GFA) is proposed to proceed via a highly unusual mechanism involving translocation of this second zinc ion to another undefined site on the protein. HMG formation is subsequently catalysed at this site before the zinc ion returns to the trigonal planar site after catalysis Paracoccus denitrificans
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.4.1.22 5,6,7,8-tetrahydromethanopterin + formaldehyde
-
 Paracoccus denitrificans 5,10-methylenetetrahydromethanopterin + H2O
-
 ?
4.4.1.22 additional information the enzyme glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans catalyzes S-hydroxymethylglutathione (HMG) formation from glutathione and formaldehyde. Enzyme GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. It is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context. NMR exchange spectroscopy is used for ligand identification. Glutathione binding structure, overview Paracoccus denitrificans ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.4.1.22 Gfa
-
 Paracoccus denitrificans
4.4.1.22 glutathione-dependent formaldehyde-activating enzyme
-
 Paracoccus denitrificans

General Information

EC Number General Information Comment Organism
4.4.1.22 additional information GFA catalysis is proposed to proceed via a highly unusual mechanism involving translocation of this second zinc ion to another undefined site on the protein. HMG formation is then catalysed at this site before the zinc ion returns to the trigonal planar site after catalysis Paracoccus denitrificans
4.4.1.22 physiological function GFA binds glutathione but does not directly catalyse S-hydroxymethylglutathione formation under standard conditions. Instead, it may be a glutathione carrier that acts to colocalise glutathione and formaldehyde in a cellular context Paracoccus denitrificans
4.4.1.22 physiological function the enzyme glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans catalyzes S-hydroxymethylglutathione (HMG) formation from glutathione and formaldehyde. HMG may then be oxidised by an alcohol dehydrogenase to form S-formylglutathione, which is further metabolised by S-formylglutathione hydrolase to give formate, thus returning GSH to the cellular pool. Enzyme GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. It is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context Paracoccus denitrificans