A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two-dimensional proton exchange NMR spectroscopy.
The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.
Goenrich
Meike
M
Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany.
Bartoschek
Stefan
S
Hagemeier
Christoph H
CH
Griesinger
Christian
C
Vorholt
Julia A
JA
D019730:Carbon-Sulfur Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D005557:Formaldehyde / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009586:Nitrogen Fixation; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000254:growth & development
D003470:Culture Media; D005557:Formaldehyde; C029469:S-hydroxymethylglutathione; D019730:Carbon-Sulfur Ligases; C447825:glutathione-dependent formaldehyde-activating enzyme; D005978:Glutathione