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Papers In Press, published online ahead of print December 10, 2001
Laboratoire de Biologie Moleculaire des Relations Plantes-Microogranismes, INRA/CNRS, Castanet-Tolosan 31326
Corresponding Author: vorholt{at}toulouse.inra.fr
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 2D proton exchange NMR spectroscopy (EXSY). The pseudo first order rates k1* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalysed reaction. At 303K and pH 6.0 k1* was found to be 0.02 s-1 for the spontaneous reaction. A ten-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 U mg-1. Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 U mg-1. The enzyme catalysing 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 catalyses 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.
J. Biol. Chem, 10.1074/jbc.C100579200
Submitted on October 5, 2001
Revised on December 3, 2001
Accepted on December 10, 2001
A glutathione-dependent formaldehyde activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via 2D proton exchange NMR spectroscopy
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