Characterization of glutathione amide reductase from Chromatium gracile - Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling
B. Vergauwen et al., Characterization of glutathione amide reductase from Chromatium gracile - Identification of a novel thiol peroxidase (Prx/Grx) fueled by glutathione amide redox cycling, J BIOL CHEM, 276(24), 2001, pp. 20890-20897
Among the Chromatiaceae, the glutathione derivative gamma -L-glutamyl-L-cys
teinylglycine amide, or glutathione amide, was reported to be present in fa
cultative aerobic as well as in strictly anaerobic species, The gene (garB)
encoding the central enzyme in glutathione amide cycling, glutathione amid
e reductase (GAR), has been isolated from Chromatium gracile, and its genom
ic organization has been examined. The garB gene is immediately preceded by
an open reading frame encoding a novel 27.5-kDa chimeric enzyme composed o
f 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 glutathion
e 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 reducta
ses 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 Michae
lis constants of 97 and 13.2 muM for glutathione amide disulfide and NADH,
respectively (in contrast to K-m 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 crystallographi
c data of the human glutathione reductase to hydrogen-bond one of the gluta
thione 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-2
24, 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 th
e newest member of the class I flavoprotein disulfide reductase family of o
xidoreductases.