Involvement of the Azotobacter vinelandii rhodanese-like protein RhdA in the glutathione regeneration pathway.
The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys(230)Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys(230) residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys(230)Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.
Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università degli Studi di Milano, Milano, Italy.
William
W
Remelli
Nicoletta
N
Guerrieri
Jennifer
J
Klodmann
Jutta
J
Papenbrock
Silvia
S
Pagani
Fabio
F
Forlani
D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005609:Free Radicals / Q000378:metabolism; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism; D062105:Molecular Docking Simulation; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*
D001426:Bacterial Proteins; D005609:Free Radicals; D008054:Lipid Peroxides; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase; D005978:Glutathione; D003545:Cysteine