Santos R, Bocquet S, Puppo A, Touati D
Laboratoire de Génétique Moléculaire des Réponses Adaptatives, Institut Jacques Monod, CNRS-Universités Paris 6 et 7, 75251 Paris Cedex 05, France.
J Bacteriol. 1999 Aug;181(15):4509-16. doi: 10.1128/JB.181.15.4509-4516.1999.
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 detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. The sodA gene 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 aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2). The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite 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 monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S. meliloti SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H(2)O(2). 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 resistance to H(2)O(2) of this unusual Fe-type SOD.
苜蓿中华根瘤菌Rm5000是一种需氧细菌，它可以自由生活在土壤中，也可以与豆科植物的根共生。在自由生活的生长条件下发现了一种可检测到的超氧化物歧化酶（SOD）。通过使用从简并引物经PCR扩增得到的sod片段作为探针，从基因组文库中分离出了相应的基因。sodA基因位于染色体上。它以单顺反子形式转录，编码一种含有200个氨基酸的蛋白质，理论分子量为22430，等电点为5.8。当该基因从合成的tac启动子而非其自身启动子表达时，苜蓿中华根瘤菌SOD可互补缺陷型大肠杆菌突变体，恢复sodA sodB recA菌株的需氧生长。氨基酸序列比对显示与含铁超氧化物歧化酶（FeSOD）有很大相似性，但该酶不会被H₂O₂灭活。纯化后的天然酶是一种二聚体蛋白，比活性为4000 U/mg。尽管其具有Fe型序列，但原子吸收光谱显示锰是其辅因子（每摩尔单体含0.75摩尔锰和0.24摩尔铁）。脱辅基酶是从苜蓿中华根瘤菌的粗提物中制备的。通过用MnCl₂或Fe(NH₄)₂(SO₄)₂透析可恢复活性，这证明了苜蓿中华根瘤菌SOD的兼性本质。用锰恢复的活性比用铁高七倍。两种重组酶都对H₂O₂有抗性。与70种FeSOD和MnSOD的序列比较表明，苜蓿中华根瘤菌SOD在特定位点含有几个非典型残基，这可能解释了这种不寻常的Fe型SOD被锰激活以及对H₂O₂抗性的原因。
