Unique Characteristics of Recombinant Hybrid Manganese Superoxide Dismutase from Staphylococcus equorum and S. saprophyticus

Abstract

A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at -20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na(+), K(+)), divalent (Ca(2+), Mg(2+)), multivalent (Mn(2+/4+), Zn(2+/4+)) cations and anions (Cl(-), SO4 (2-)) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO4, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO4, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.

Activity of Superoxide Dismutase Mimic of [Mn(salen)OAc] Complex Compound Non-enzymatically in Vitro Through Riboflavin Photoreduction

“…•-species sources which often used are derived from flavin photoreduction (Kostyuk, Potapovich, Kostyuk, & Cherian, 2007;Retnoningrum et al, 2016), organic compound autooxidation (Li, 2012) and gamma irradiation (Barnese, Gralla, Cabelli, & Valentine, 2008). The nonenzymatic method through riboflavin photoreduction is currently used because it is easier to use, relatively cheaper, and accurate.…”

“…The nonenzymatic method through riboflavin photoreduction is currently used because it is easier to use, relatively cheaper, and accurate. (Retnoningrum et al, 2016) and SOD-manganese from Staphylococcus equorum (Indrayati, Asyarie, Suciati, & Retnoningrum, 2014). However, there were only found 9 SOD mimics of manganese complex compound whose activities were determined by this rb-NBT non enzymatic method, those are: 1 complex of manganese(II)-bovine serum albumin (Yan et al, 2016) , 2011).…”

“…In the standard mixture and its blank (blank 2), methanol solvent was added instead of the sample solution. The difference of absorbance between standard and samples (without and with riboflavin, respectively) was multiplied by 100% to obtain % inhibition of each various sample concentration against NBT (Retnoningrum et al, 2016).…”

The first crystal structure of manganese superoxide dismutase from the genusStaphylococcus

“…In initial work on S. equorum MnSOD only a partial nucleic acid sequence was elucidated; the full nucleotide sequence of the enzyme was reconstructed by insertion of the N-and C-termini of the gene encoding S. saprophyticus MnSOD (Retnoningrum et al, 2016). Therefore, the enzyme has previously been referred to as a hybrid of the MnSODs from S. equorum and S. saprophyticus (Retnoningrum et al, 2016). Subsequently, the amino-acid sequence was found to be almost identical to the more recently reported MnSOD sequence from S. equorum (GenBank WP_046465435.1) with the only substitution being at Asp13Arg.…”

“…For example, S. equorum MnSOD is still active at 353 K (Retnoningrum et al, 2016), at which temperature B. subtilis MnSOD is completely inactivated (Wang et al, 2014). S. equorum MnSOD is also more resistant to inactivation by urea or guanidinium chloride (chaotropic agents), by sodium dodecyl sulfate (a detergent) or by -mercaptoethanol (a reducing agent) than B. subtilis MnSOD (Retnoningrum et al, 2016). These findings suggest that the B. subtilis MnSOD structure may be not representative of S. equorum MnSOD.…”