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Originally published In Press as doi:10.1074/jbc.C100579200 on December 10, 2001

J. Biol. Chem., Vol. 277, Issue 5, 3069-3072, February 1, 2002
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ACCELERATED PUBLICATION
A Glutathione-dependent Formaldehyde-activating Enzyme (Gfa) from Paracoccus denitrificans Detected and Purified via Two-dimensional Proton Exchange NMR Spectroscopy*,

Meike GoenrichDagger §, Stefan BartoschekDagger §||, Christoph H. HagemeierDagger **, Christian Griesinger, and Julia A. VorholtDagger Dagger Dagger

From the Dagger  Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany, the  Institut für Organische Chemie der Universität Frankfurt, Marie-Curie-Strasse 11, 60439 Frankfurt a.M., Germany, and the Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany

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 k1* 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 k1* 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.

* This work was supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. All NMR measurements were conducted at the European Large Scale Facility for Biomolecular NMR (ERBCT95-0034) at the University of Frankfurt.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 2 and 3.

§ These authors contributed equally to this work.

|| Supported by a Kekulé stipend of the Fonds der Chemischen Industrie.

** Supported by the Peter and Traudl Engelhorn Stiftung.

Dagger Dagger To whom correspondence should be addressed: INRA/CNRS, BP27 Chemin de Borde Rouge, 31326 Castanet-Tolosan, France. Tel.: 33-5-61-28-54-58; Fax: 33-5-61-28-50-61; E-mail: vorholt@toulouse.inra.fr.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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