Superoxide Dismutases from Hyperthermophilic Archaea
Shigeyuki YAMANO*a, Yoshihiko SAKOb, Norimichi NOMURAb, and Tadashi MARUYAMAa
a Marine Biotechnology Institute, 1900 Sodeshi-cho, Shimizu 424, Japan
b Department of Applied Bioscience, Kyoto University, Kitashirakawa-oiwake-chc, Kyoto 606-01, Japan
Superoxide dismutase (SOD) catalyzes dismutation of superoxide anion into oxygen and hydrogen peroxide. These enzymes play an important role in protection of cell mechanisms from oxidative damage. The SOD enzyme family is classified into three metalloprotein groups according to the metal cofactor: copper and zinc containing SOD, manganese containing SOD and iron containing SOD. The latter two groups are assumed to have a common evolutionary origin because of the high similarity in an amino acid sequence and three-dimensional protein structure. Hyperthermophilic Bacteria and Archaea occupy all the deepest and shortest branches of the phylogenetic tree. Therefore, they may retain primitive characteristics close to those of progenotes. Because most of hyperthermophiles belong to Archaea and several to Eubacteria, it is interesting to study the character of hyperthermophilic archaeal SODs from an evolutionary point of view. We cloned SOD genes from aerobic hyperthermophilic archaea, Aeropyrum pernix and Sulfolobus solfataricus by using PCR products as a probe. Primers designed from conserved regions of Mn or Fe containing SODs were used for PCR. The deduced amino acid sequences of these genes show similarities to known SODs from archaea which are not hyperthermophiles. These SOD genes were highly expressed in E. coli cells and SOD proteins were purified. The proteins are highly thermostable and no loss of activity was observed after incubation at 99℃ for 30 minutes. In E. coli cells, both Mn and Fe were incorporated into each respective SOD, though metal contents were variable. We studied relationships between metal content and activities of SOD. The activity of S. solfataricus SOD depended on Fe content, and Mn did not appear to contribute to activity. On the other hand, metal-reconstitution experiments of A. pernix SOD showed that the Mn-substituted form was more active than the Fe-substituted one. Thus A. pernix SOD is a "cambialistic" one which is active with either Fe or Mn as a cofactor. Azide is a competitive inhibitor of all known SODS. However, azide did not inhibit S. solfataricus SOD nor the Mn-substituted form of A. pernix SOD up to 400 mM. Azide of 41 mM did cause 50% inhibition of the Fe-substituted form of A. pernix SOD. It may be difficult to inhibit hyperthermophilic archaeal SODs by azide. Hydrogen peroxide inactivated the Fe-substituted form of A. pernix SOD but not the Mn-substituted form. Inactivation of Fe-SOD is supposed to be accompanied by decomposition of the tryptophan residue near the active site. We presume that inactivation of Fe-substituted A. pernix SOD by hydrogen peroxide is caused in the same manner as other Fe-SODs.
