Regulation of the properties of superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans by iron- and manganese-bound co-factor

Abstract

Streptococcus mutans, the main pathogen involved in the development of dental caries, is an aerotolerant microorganism. The bacterium lacks cytochromes and catalase, but possesses other antioxidant enzymes, such as superoxide dismutase (SmSOD). Previous researches suggested that SmSOD belongs to the 'cambialistic' group, functioning with Fe or Mn in the active site. A recombinant SmSOD (rSmSOD) with a His-tail has been produced and characterised. Studies on metal uptake and exchange proved that rSmSOD binds either Fe or Mn as a metal co-factor, even though with a consistent preference for Fe accommodation. The analysis of several enzyme samples with different values of the Mn/Fe ratio in the active site proved that the type of metal is crucial for the regulation of the activity of rSmSOD. Indeed, differently from the significant preference for Fe displayed by the enzyme in the binding reaction, its Mn-form was 71-fold more active compared to the Fe-form. The rSmSOD was endowed with a significant thermostability, its half-inactivation occurring after 10 min exposure at 71 or 73 degrees C, depending on the bound metal. Moreover, the enthalpic and entropic contribution to the heat inactivation process of rSmSOD were strongly regulated by the Mn content of the enzyme. The effect of typical inhibitors/inactivators has been investigated. rSmSOD was inhibited by sodium azide, and its sensitivity increased in the presence of higher Mn levels. Concerning two physiological inactivators, the enzyme displayed a different behaviour, being quite resistant to hydrogen peroxide and significantly sensitive to sodium peroxynitrite. Furthermore, the Mn co-factor had an amplifying role in the regulation of this different sensitivity. These results confirm the cambialistic nature of SmSOD and prove that its properties are regulated by the different metal content. The adaptative response of S. mutans during its aerobic exposure in the oral cavity could involve a different metal uptake by SmSOD.

Novel Antibiofilm Chemotherapy Targets Exopolysaccharide Synthesis and Stress Tolerance in Streptococcus mutans To Modulate Virulence Expression In Vivo

“…SloA is required for virulence and oxidative stress tolerance by S. mutans (46,52), whereas CopY may play a role in biofilm detachment (42). Metal accumulation in bacteria is not only important for its potential antioxidant benefit, but metals also frequently serve as cofactors for critical enzymatic and metabolic reactions, as is the case for superoxide dismutase (SodA) (13,44,53). SodA is an established virulence factor that reduces superoxide and is essential in vivo, the repression/dysfunction of which may be linked to repression of sloA, since manganese and iron are key cofactors for this enzyme (13).…”

“…Metal accumulation in bacteria is not only important for its potential antioxidant benefit, but metals also frequently serve as cofactors for critical enzymatic and metabolic reactions, as is the case for superoxide dismutase (SodA) (13,44,53). SodA is an established virulence factor that reduces superoxide and is essential in vivo, the repression/dysfunction of which may be linked to repression of sloA, since manganese and iron are key cofactors for this enzyme (13). Upregulation of lrgA and/or changes in the expression of fatty acid synthesis genes (e.g., upregulation of fabK) following CT treatment may be another indicator that the combination therapy is compromising the fitness of S. mutans, since it may correlate with alterations in the membrane physiology.…”

Properties of the endogenous components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125

“…In the case of PhTrxR, the half-inactivation temperature of the flavoenzyme (60°C) is well above the maximum growth temperature of 20°C tolerated by P. haloplanktis. The energetics of the heat inactivation process of PhTrxR was also investigated and the calculated value of E a (154 kJ mol -1 ), although higher than that reported for polynucleotide phosphorylase (96.7 kJ mol -1 ), another enzyme isolated from P. haloplanktis ), still reflects the psychrophilic origin of the source, as it is lower than the values usually reported for mesophilic and thermophilic enzymes, showing an average E a value of 278 ± 60 kJ mol -1 (Masullo et al 1993;Grimaldi et al 2008;Castellano et al 2009;De Vendittis et al 2010). Furthermore, since the denaturation profile of the flavoenzyme determined through fluorescence-melting curves is roughly similar to the heat inactivation profile, it is possible to conclude that heat inactivation and protein denaturation occur in the same temperature interval.…”

Properties of a Putative Cambialistic Superoxide Dismutase from the Aerotolerant Bacterium Streptococcus thermophilus Strain LMG 18311

“…The enzyme, whose expression depended on the growth phase, was insensitive to cyanide or H 2 O 2 treatment, a behaviour suggesting that SOD from S. thermophilus (StSOD) was a Mncontaining enzyme. However, no direct metal detection was presented to rule out the possibility of a cambialistic StSOD, as already demonstrated for the corresponding enzyme from the taxonomically related source S. mutans (SmSOD) [24,27], nor the characterization of the biochemical properties of the purified enzyme was carried out. Another unresolved question was the size of SOD from the S. thermophilus strain LMG 18311, as predicted from its putative encoding gene, because it presented a N-terminal extension absent in other bacterial SODs [7].…”

“…The presence of Fe or Mn in the active site of the enzyme is dictated by specific metal requirements and/or physiological conditions; moreover, the exchange between these two metal ions usually leads to loss of enzyme activity [17,[20][21]. On the other hand, some prokaryotes with facultative anaerobic growth conditions possess a cambialistic SOD belonging to the same group of this ubiquitous SOD family; the enzyme, able to bind and exchange Fe or Mn in the active site, displays substantial though different activity with each metal [22][23][24][25][26][27]. Previous reports described the presence of a single SOD in S. thermophilus strain AO54 [28] and analysed the functions of the corresponding gene [29].…”