Abstract
The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg−1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H2O2. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by β-naphthoquinone-4-sulfonic acid, ρ-diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.
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References
Agostini HJ, Carroll JD, Minton KW (1996) Identification and characterization of uvrA, a DNA repair gene of Deinococcus radiodurans. J Bacteriol 178:759–765
An SS, Kim YM (1997) Purification and characterization of a manganese-containing superoxide dismutase from a carboxydobacterium Pseudomonas carboxydohydrogen. Mol Cells 7:730–737
Battista JR, Earl AM, Park MJ (1999) Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol 7:362–365
Bauche C, Laval J (1999) Repair of oxidized bases in the extremely radiation-resistant bacterium Deinococcus radiodurans. J Bacteriol 181:262–269
Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287
Bollag DM, Edelstein SJ (1996) Protein methods, 3rd edn. Wiley, New York
Borgstahl GE, Parge HE, Hickey MJ, Beyer WFJ, Hallewell RA, Tainer JA (1992) The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell 71:107–118
Carroll JD, Daly MJ, Minton KW (1996) Expression of recA in Deinococcus radiodurans. J Bacteriol 178:130–135
Chou FI, Tan ST (1990) Manganese (II) induces cell division and increases in superoxide dismutase and catalase activities in an aging deinococcal culture. J Bacteriol 172:2029–2035
Evans DM, Moseley BE (1983) Roles of the uvsC, uvsD, uvsE, and mtcA genes in the two pyrimidine dimer excision repair pathways of Deinococcus radiodurans. J Bacteriol 156:576–583
Gersten DM (1996) Gel electrophoresis: proteins. In: Rickwood D (ed) Essential techniques series. Wiley, New York
Gutman PD, Carroll JD, Masters CI, Minton KW (1994) Sequencing, targeted mutagenesis and expression of recA gene required for the extreme radioresistance of Deinococcus radiodurans. Gene 141:31–37
Hakamada Y, Koike K, Kobayashi T, Ito S (1997) Purification and properties of mangano-superoxide dismutase from a strain of alkalophilic Bacillus. Extremophiles 1:74–78
Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine, 3rd edn. Oxford University Press, Oxford, pp 105–245
Inaoka T, Matsumura Y, Tsuchido T (1998) Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis. J Bacteriol 180:3697–3703
Jung SY, Lee SL, Lee TH (1993) Purification and characterization of superoxide dismutase from Rhodotorula glutinis K-24. Kor J Microbiol 31:573–578
Kim J, Sharma AK, Abbott SN, Wood EA, Dwyer DW, Jambura A, Minton KW, Inman RB, Daly MJ, Cox MM (2002) RecA protein from the extremely radioresistant bacterium Deinococcus radiorurans: expression, purification and characterization. J Bacteriol 184:1649–1660
Lowry OH, Rosebrough NJ, Farr AC, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Makarova KS, Aravind L, Wolf YI, Tatusov RL, Minton KW, Koonin EV, Daly MJ (2001) Genome of the extremely radiation resistant bacteria, Deinococcus radiodurans, viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 65:44–79
Marklund S, Marklund G (1974) Assay of SOD by pyrogallol autooxidation. Eur J Biochem 47:469–474
Meier B, Parak F, Desideri A, Rotilio G (1997) Comparative stability studies on the iron and manganese forms of the cambialistic superoxide dismutase from Propionibacterium shermanii. FEBS Lett 414:122–124
Minton KW (1996) Repair of ionizing radiation damage in the radiation resistant bacterium Deinococcus radiodurans. Mutant Res 363:1–7
Misra HP, Fridovich I (1978) Inhibition of superoxide dismutases by azide. Arch Biochem Biophys 189:317–322
Müller DJ, Engel WA (1996) Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy. J Bacteriol 178:3025–3030
Murray RGE (1986) Family II, Deinococcaceae. In: Buchanan RE, Gibbons NE (eds) Bergey’s manual of systematic bacteriology. Williams & Wilkins, Baltimore, pp 1035–1043
Osatomi K, Masuda Y, Hara K, Ishihara T (2001) Purification, N-terminal amino acid sequence, and some properties of Cu, Zn-superoxide dismutase from Japanese flounder (Paralichthys olivaceus) hepato-pancreas. Comp Biochem Phys 128:751–760
Öztürk-Ürek R, Tarhan L (2001) Purification and characterization of superoxide dismutase from chicken liver. Comp Biochem Phys 128:205–212
Öztürk-Ürek R, Bozkaya LA, Atav E, Saglam N, Tarhan L (1999) Purification and characterization of superoxide dismutase from Phanerchaete chrysosporium. Enzyme Microbiol Tech 25:392–399
Pagani S, Colnaghi R, Palagi A, Negri A (1995) Purification and characterization of an iron superoxide dismutase from the nitrogen-fixing Azotobacter vinelandii. FEBS Lett 357:79–82
Price NC, Stevens L (1988) Fundamentals of enzymology, 2nd edn. Oxford Science, Oxford
Righetti P, Drysdale JW (1971) Isoelectric focusing in polyacrylamide gels. Biochem Biophys Acta 236:17–28
Soung NK, Lee YN (2000) Iso-catalase profiles of Deinococcus spp. J Biochem Mol Biol 33:412–416
Uchica K, Kawakishi S (1994) Identification of oxidized histidine generated at the active site of CuZnSOD exposed to H2O2. J Biol Chem 269:2405–2410
Valderas MW, Hart ME (2001) Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus. J Bacterial 183:3399–3407
Youn HD, Kim EJ, Roe JH, Hah YC, Kang SO (1996). A novel nickel-containing superoxide dismutase from Streptomyces spp. Biochem J. 318:889–896
Yun EJ, Lee YN (2000) Production of two different catalase-peroxidases by Deinococcus radiophilus. FEMS Microbiol Lett 184:155–159
Yun YS, Lee YN (2001) Superoxide dismutase profiles in the mesophilic Deinococcus species. J Microbiol 39:232–235
Yun YS, Lee YN (2003) Production superoxide dismutase by Deinococcus radiophilus. J Biochem Mol Biol 36:282–287
Acknowledgements
This work was supported by a grant (ROS-2001-000-00320-0) from the Basic Research Program of the Korea Science and Engineering Foundation.
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Yun, Y.S., Lee, Y.N. Purification and some properties of superoxide dismutase from Deinococcus radiophilus, the UV-resistant bacterium. Extremophiles 8, 237–242 (2004). https://doi.org/10.1007/s00792-004-0383-6
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DOI: https://doi.org/10.1007/s00792-004-0383-6