Superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans

Streptococcus mutans is an eubacterial pathogenic microorganism involved in the development of dental caries, growing either in aerobic or anaerobic conditions. The bacterium lacks cytochromes and catalase, but possesses other anti-oxidant enzymes involved in the control of the intracellular redox potential, such as superoxide dismutase (SOD), the key enzyme scavenging the toxic superoxide anions. Previous researches suggested that SOD from S. mutans (SmSOD) belongs to the cambialistic group, functioning with Fe or Mn in the active site; this feature is probably related to the adaptation of this microorganism to different growth conditions. Recently, the genome of S. mutans has been sequenced, and one SOD gene has been identified. SmSOD was heterologously produced in Escherichia coli, with its C-terminal extremity fused to a His-tag. This recombinant form of the enzyme (rSmSOD) had a specific activity of 1150 U/mg and contained both Fe and Mn (0.45 and 0.07 atoms/subunit, respectively). To improve its metal content, rSmSOD was incubated in the presence of Mn or Fe ions and, after extensive dialysis, metal content and specific activity were redetermined. The amount of Mn in the sample treated with Mn (SmSOD–Mn) raised to 0.21 atoms/subunit, whereas the Fe content remained unchanged. Moreover, the specific activity of SmSOD–Mn improved to 3500 U/mg. Vice versa, in the sample treated with Fe (SmSOD–Fe), the Fe content reached 0.52 atoms/subunit at the expense of Mn; the specific activity of SmSOD-Fe slightly decreased to 1050 U/mg. rSmSOD was half-inactivated after 10 min exposure at 68C. The effect of typical inhibitors/inactivators of Fe- and Mn-SODs was investigated, in terms of a possible regulation of the sensitivity by samples with different metal contents. Sodium azide functioned as a weak inhibitor of SmSOD, the lowest resistance being displayed by SmSOD-Fe. Hydrogen peroxide caused a modest inactivation on either untreated rSmSOD or SmSODFe, whereas it was almost ineffective on SmSOD–Mn. Sodium peroxynitrite provoked a significant inactivation of SmSOD–Mn; the untreated rSmSOD and even more SmSOD–Fe were more resistant to peroxynitrite inactivation. These results confirm the cambialistic nature of SmSOD and prove its regulation by different metal contents. Therefore, the adaptative response of S. mutans during the aerobic exposure in the oral cavity could involve a different metal uptake of SmSOD.