Superoxide dismutase from the extremely halophilic archaebacterium Halobacterium cutirubrum

Abstract

Halobacterium cutirubrum, a member of the archaebacteria, contains one superoxide dismutase (EC 1.15.1.1). This enzyme functions in the high-ionic-strength intracellular environment and protects the organism against the toxic effects of the superoxide anion. The enzyme has been purified to about 90% homogeneity by a four-step procedure which never removes it from conditions of high ionic strength. The subunits of the purified enzyme have a molecular weight of 25,000 and are possibly in tetrameric association. Halobacteria are aerobic, mesophilic heterotrophs which inhabit brines containing, ideally, 4 to 5 M NaCl (8, 16). As members of the archaebacteria, they are only distantly related to eubacteria and eucaryotes (33). The halobacteria are phylogenetically related to the methanogenic archaebacteria and are believed to have evolved from anaerobic, nonhalophilic ancestors (26, 33). Two major steps were required for this evolution. One was the adaptation to high salt concentrations; this was achieved by raising the intracellular salt concentration to near saturation and altering enzyme structures to function in this environment (3,14,15). The second was the adaptation to oxygen and aerobic respiration, which necessitated the development of mechanisms to protect against toxic oxygen radicals such as the superoxide anion.One key enzyme involved in this protection is superoxide dismutase (SOD) (9,20). It catalyzes the dismutation of superoxide to peroxide and molecular oxygen (6). On the basis of metal content, three types of SOD have been described: copper-zinc SOD in eucaryotic cytoplasm, manganese SOD in eubacteria and mitochondria, and iron SOD in eubacteria (29). To date, SOD has been isolated from two species of archaebacteria, Methanobacterium bryantii (11), an anaerobe, and Thermoplasma acidophilum (27) Cell culture. H. cutirubrum was grown to mid-log phase (A460 = 1.0) in rich medium as described previously (12).SOD purification. Hydroxylapatite chromatography and gel filtration were performed at 20°C. All other steps were performed at 4°C, and all buffers contained 1 mM 2-mercaptoethanol. Cells from 4 liters of culture were pelleted at 4,000 x g for 20 min and then suspended in 40 ml of 3.0 M ammonium sulfate-50 mM sodium phosphate (pH 7.0). The cells were lysed by five 2-min sonications (0.15 relative output in a Fisher model 150 dismembrator). The lysate was centrifuged at 44,000 x g for 12 h. The supernatant was dialyzed against lysis buffer for 4 h and recentrifuged at 114,000 x g for 2 h. The supernatant was adjusted to 2.5 M ammonium sulfate and adsorbed onto a column (60 by 2.6 cm) of Sepharose CL-4B which had been equilibrated with 2.25 M ammonium sulfate-50 mM sodium phosphate (pH 7.0). The column was washed with 700 ml of buffer, and the SOD activity was then eluted with a 1.3-liter decreasing linear ammonium sulfate gradient (2.25 to 1.40 M in phosphate buffer; Fig. 1). Fractions containing SOD activity were pooled and applied to a column (35 by 2.6 cm) of DEAESepharose CL-6B which had been equi...

Characterization of paralogous and orthologous members of the superoxide dismutase gene family from genera of the halophilic archaebacteria

“…This sequence forms a complementary structure with the highly conserved pyrimidine-rich sequence at the 3' end of 16S rRNA (35 (13,22). Primer extension, S1 nuclease protection and Northern hybridization experiments demonstrate that the amount of sod mRNA increases concomitantly with the increase in enzyme activity (23 Notably, two of the blocks of conservation within this region are the box A promoter element (-44 to -39) and the potential ribosome binding sequence (-14 to -10).…”

“…Molecular length markers were generated by using the same 5'-end-labeled oligonucleotides as primers in a DNA sequencing reaction using appropriate template DNA. Enzyme activity assay. SOD activity was assayed as previously described (20,22 [33].) Genomic DNA from Haloferax volcandi and the DNAs from these two cosmids were digested with Sau3AI and reprobed with the 1.1-kbp sod fragment from H. cutirubrum (Fig.…”

“…The sod gene encodes a protein with SOD activity; this protein has been purified to near homogeneity and has been well characterized (22). The slg gene is actively transcribed, but its protein product has not been identified.…”

Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium

“…These data are consistent with reports that various halobacterial proteins contain an excess of acidic amino acids (for reviews, see references 18 and 31). The optimum salt concentrations for enzymatic activities for H. cutirubrum are around 1 M for photolyase (A. P. M. Eker, personal communication) and 2 M for SOD (21). The higher acidity of these enzymes in Halobacterium species must have resulted from an adaptation to the extremely high salt environments they inhabit.…”

Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria

“…SOD activity has been detected in at least two representatives of the ancestral methanogen group and in numerous halophilic species (7,10,25). The SOD enzyme from both Halobacterium cutirubrum and its immediate relative, H. halobium, has been purified and extensively characterized; the enzyme contains Mn as a metal ion cofactor and is related to the Mnor Fe-type enzymes of eubacteria (13,19).The sod gene, encoding the authentic SOD protein, was cloned from the genome ofH. cutirubrum, and its expression was shown to be elevated in the presence of paraquat, a compound that generates superoxide radicals (14, 15).…”