Characterization of an atypical superoxide dismutase from Sinorhizobium meliloti

Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single det ectable superoxide dismutase (SOD) was found in free-living growth conditio ns. The corresponding gene was isolated from a genomic library by using a s ad fragment amplified by PCR from degenerate primers as a probe. The sodA g ene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M-r of 22,430 and pI of 5.8. S. meliloti SOD complemented a deficient E. coli mutant, restoring ae robic growth of a sodA sodB recA strain, when the gene was expressed from t he synthetic tac promoter but not from its own promoter. Amino acid sequenc e alignment showed great similarity with Fe-containing SODs (FeSODs), but t he enzyme was not inactivated by H2O2. The native enzyme was purified and f ound to be a dimeric protein, with a specific activity of 4,000 U/mg. Despi te its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of mon omer). The apoenzyme was prepared from crude extracts of S. meliloti, Activ ity was restored by dialysis against either MnCl2 or Fe(NH4)(2)(SO4)(2), de monstrating the cambialistic nature of the S. meliloti SOD. The recovered a ctivity with manganese was sevenfold higher than with iron. Both reconstitu ted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resi stance to H2O2 of this unusual Fe-type SOD.