Two superoxide dismutases from TnOtchr are involved in detoxification of reactive oxygen species induced by chromate

Rita Branco, Paula V. Morais

Superoxide dismutases (SOD) have been reported as the most relevant bacterial enzymes involved in cells protection from reactive oxygen species (ROS). These toxic species are often the product of heavy metal stress. Two genes, chrC and chrF, from TnOtchr genetic determinant of strain Ochrobactrum tritici 5bvl1 were cloned in Escherichia coli in order to overexpress the respective proteins. Both proteins were purified and characterized as superoxide dismutases. ChrC was confirmed as being a Fe-SOD, and the enzymatic activity of the ChrF, not inhibited by hydrogen peroxide or potassium cyanide, suggested its inclusion in the Mn-SOD family. This identification was supported by chemical quantification of total metal content in purified enzyme. Both enzymes showed a maximum activity between pH 7.2-7.5. ChrF retained nearly full activity over a broader range of pH and was slightly more thermostable than ChrC. The genes encoding these enzymes in strain O. tritici 5bvl1 were inactivated, developing single and double mutants, to understand the contribution of these enzymes in detoxification mechanism of reactive oxygen species induced by chromate. During chromate stress, assays using fluorescent dyes indicated an increase of these toxic compounds in chrC, chrF and chrC/chrF mutant cells. In spite of the multiple genes coding for putative superoxide dismutase enzymes detected in the genome of O. tritici 5bvl1, the ChrC and ChrF might help the strain to decrease the levels of reactive oxygen species in cells.