Purification and some properties of superoxide dismutase from Deinococcus radiophilus , the UV-resistant bacterium

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 degrees C, but a linear reduction in activity above 50 degrees C. Inhibition of the purified SOD activity by beta-naphthoquinone-4-sulfonic acid, rho-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.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

“…The manganese-containing enzyme superoxide dismutase from yeast showed progressively less activity at pHs above 7.8 [50]. Superoxide dismutase from Deinococcus radiophilus was stable up to 50 • C, but was, however, highly unstable at pHs below 5.0 [51]. The iron-and manganese-containing superoxide dismutase from Methylobacillus [52], exhibits 66% activity at pH 5.0, but it is completely inactivated after 4 min incubation at 90 • C. Two manganese-containing superoxide dismutases from thermophilic fungi Chaetomium thermophilum and Thermomyces lanuginosus remain active up to 60 and 70 • C (thermal stability), respectively, and lose the activity at pH below 5.0 [8].…”

Characterization of Deinococcus radiophilus thioredoxin reductase active with both NADH and NADPH

“…The cell-free extracts were obtained from sonicated-preparations of the cells at 4°C for 60 min (20 sec pulse on and 40 sec pulse off) with a sonic dismembrator (Fisher sonic dismembrator, Fisher Scientific Co., USA) and then centrifugation at 12,000 rpm for 20 min (Yun and Lee, 2004).…”

Iso-superoxide dismutase in Deinococcus grandis, a UV resistant bacterium

“…The presence of iso-SODs in living organisms, including eukaryotes and procaryotes has also been exclusively reported (Benov and Fridovich, 1994;Sadosky et al, 1994;Fridovich, 1995;Schnell and Steinman, 1995;St. John and Steinman, 1996;Li et al, 2002;Yun and Lee, 2004). When production studies regarding changes of D. grandis iso-SODs in response to exogenous oxidative stress, such as superoxide-and peroxide-treatment or UV irradiation were conducted, the production pattern of SODs was somewhat dissimilar.…”

“…Cyanide is a reversible inhibitor of SODs that exerts a dose-dependent effect whereas H2O2 is an irreversible inhibitor (Fridovich, 1995). It is known that MnSOD is insensitive to not only cyanide (CN -) but also H2O2, whereas CuZnSOD is sensitive to both chemicals, and FeSOD is sensitive to H2O2 but not to CN - (Kroll et al, 1991;Yun and Lee, 2004). When PAGE gels resolved proteins in cell-free extract prepared from the stationary culture of D. grandis were treated with either H2O2 or KCN and followed the SOD activity staining, activity of SOD-1 was not affected by such treatments, suggesting that SOD-1 is MnSOD.…”

“…The SOD bands on the gel were analyzed by densitometry using a Kodak eletrophoresis documentation and analysis system (Kodak ID Image Analysis, Japan). The sensitivity of the SODs on the gel to chemicals was evaluated by soaking the gel in 50 mM potassium phosphate buffer (pH 7.8) containing either 20 mM H2O2 or 10 mM KCN for 60 min at room temperature prior to activity staining of the SOD (Kroll et al, 1991;Yun and Lee, 2004).…”