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Originally published In Press as doi:10.1074/jbc.M102026200 on April 3, 2001

J. Biol. Chem., Vol. 276, Issue 24, 20890-20897, June 15, 2001
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Characterization of Glutathione Amide Reductase from Chromatium gracile
IDENTIFICATION OF A NOVEL THIOL PEROXIDASE (Prx/Grx) FUELED BY GLUTATHIONE AMIDE REDOX CYCLING*

Bjorn VergauwenDagger , Frederik PauwelsDagger , Françoise Jacquemotte§, Terrance E. Meyer, Michael A. Cusanovich, Robert G. Bartsch||, and Jozef J. Van BeeumenDagger **

From the Dagger  Laboratory of Protein Biochemistry and Protein Engineering, Gent University, 9000 Gent, Belgium, the § Department Substances Naturelles, Haute Ecole Luria de Brouckère, Institut Meurice, 1070 Bruxelles, Belgique, the  Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, and the || Department of Chemistry, University of California, San Diego, La Jolla, California 92093

Among the Chromatiaceae, the glutathione derivative gamma -L-glutamyl-L-cysteinylglycine amide, or glutathione amide, was reported to be present in facultative aerobic as well as in strictly anaerobic species. The gene (garB) encoding the central enzyme in glutathione amide cycling, glutathione amide reductase (GAR), has been isolated from Chromatium gracile, and its genomic organization has been examined. The garB gene is immediately preceded by an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed of one N-terminal peroxiredoxin-like domain followed by a glutaredoxin-like C terminus. The 27.5-kDa enzyme was established in vitro to be a glutathione amide-dependent peroxidase, being the first example of a prokaryotic low molecular mass thiol-dependent peroxidase. Amino acid sequence alignment of GAR with the functionally homologous glutathione and trypanothione reductases emphasizes the conservation of the catalytically important redox-active disulfide and of regions involved in binding the FAD prosthetic group and the substrates glutathione amide disulfide and NADH. By establishing Michaelis constants of 97 and 13.2 µM for glutathione amide disulfide and NADH, respectively (in contrast to Km values of 6.9 mM for glutathione disulfide and 1.98 mM for NADPH), the exclusive substrate specificities of GAR have been documented. Specificity for the amidated disulfide cofactor partly can be explained by the substitution of Arg-37, shown by x-ray crystallographic data of the human glutathione reductase to hydrogen-bond one of the glutathione glycyl carboxylates, by the negatively charged Glu-21. On the other hand, the preference for the unusual electron donor, to some extent, has to rely on the substitution of the basic residues Arg-218, His-219, and Arg-224, which have been shown to interact in the human enzyme with the NADPH 2'-phosphate group, by Leu-197, Glu-198, and Phe-203. We suggest GAR to be the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.

* This work was supported by the Fund for Scientific Research-Flanders Grant 3G003601 and the Bijzonder Onderzoeksfonds of the Gent University Grant 12050198.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.

** To whom correspondence should be addressed. Fax: 32-9-264-53-38; E-mail: Jozef.vanbeeumen@rug.ac.be.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.