Literature summary for 4.4.1.22 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 .

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Metals/Ions	Comment	Organism	Structure
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)

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

Organism

Organism	UniProt	Comment	Textmining
Paracoccus denitrificans	Q51669	-	-

Purification (Commentary)

Purification (Comment)	Organism
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

Reaction	Comment	Organism	Reaction ID
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
5,6,7,8-tetrahydromethanopterin + formaldehyde	-	Paracoccus denitrificans	5,10-methylenetetrahydromethanopterin + H2O	-	?
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

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

General Information

General Information	Comment	Organism
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
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
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