Streptococcus mutans is an eubacterial pathogenic micro-organism involved in the development of dental caries. It grows either in aerobic or anaerobic conditions, even though its energetic metabolism is anaerobic, as the ATP synthesis almost exclusively depends on glycolysis. Because of a missing aerobic respiration, the bacterium lacks cytochromes and enzyme systems involved in the control of oxidative stress, such as catalase. However, the bacterium possesses superoxide dismutase (SOD), the key enzyme involved in scavenging superoxide anions. The previous studies did not clearly indicate whether such SOD belongs to the cambialistic SOD family, functioning with iron or manganese, a feature possibly related to the adaptation at different growth conditions tolerated by this micro-organism. Recently, the genome of Streptococcus mutans has been sequenced (1), and besides the SOD gene, other genes involved in the control of the intracellular redox potential were identified, namely thioredoxin (Trx) and thioredoxin reductase (TrxR). The strategy followed for the characterization of these enzyme systems involved the heterologous production of recombinant forms of SOD, Trx and TrxR from S. mutans. To this aim, the coding sequences of the corresponding genes were amplified by PCR from the genome of S. mutans UA159 and then cloned into a pET expression vector. The C-terminal residue of each gene product was fused to an exa-His-tag for the purification of the heterologous protein through Ni-Agarose affinity chromatography. The first recombinant enzyme obtained with this expression system was SOD from S. mutans (SmSOD). In particular, 20 mg of pure recombinant SmSOD were obtained from 1 L of bacterial culture. The recombinant enzyme was purified in an active form, as evaluated by its ability to inhibit the citochrome c reduction using the xanthine oxidase method for measuring SOD activity. The molecular and biochemical characterization of SmSOD is in progress and involves the determination of its metal content (iron and/or manganese), as well as the effect of either typical SOD inhibitors or compounds used in the treatment of pathologies of the oral cavity.
Properties of some enzyme systems involved in the control of the redox potential of Streptococcus mutans, a dental caries pathogenic microorganism
AMATO, Massimo;
2007-01-01
Abstract
Streptococcus mutans is an eubacterial pathogenic micro-organism involved in the development of dental caries. It grows either in aerobic or anaerobic conditions, even though its energetic metabolism is anaerobic, as the ATP synthesis almost exclusively depends on glycolysis. Because of a missing aerobic respiration, the bacterium lacks cytochromes and enzyme systems involved in the control of oxidative stress, such as catalase. However, the bacterium possesses superoxide dismutase (SOD), the key enzyme involved in scavenging superoxide anions. The previous studies did not clearly indicate whether such SOD belongs to the cambialistic SOD family, functioning with iron or manganese, a feature possibly related to the adaptation at different growth conditions tolerated by this micro-organism. Recently, the genome of Streptococcus mutans has been sequenced (1), and besides the SOD gene, other genes involved in the control of the intracellular redox potential were identified, namely thioredoxin (Trx) and thioredoxin reductase (TrxR). The strategy followed for the characterization of these enzyme systems involved the heterologous production of recombinant forms of SOD, Trx and TrxR from S. mutans. To this aim, the coding sequences of the corresponding genes were amplified by PCR from the genome of S. mutans UA159 and then cloned into a pET expression vector. The C-terminal residue of each gene product was fused to an exa-His-tag for the purification of the heterologous protein through Ni-Agarose affinity chromatography. The first recombinant enzyme obtained with this expression system was SOD from S. mutans (SmSOD). In particular, 20 mg of pure recombinant SmSOD were obtained from 1 L of bacterial culture. The recombinant enzyme was purified in an active form, as evaluated by its ability to inhibit the citochrome c reduction using the xanthine oxidase method for measuring SOD activity. The molecular and biochemical characterization of SmSOD is in progress and involves the determination of its metal content (iron and/or manganese), as well as the effect of either typical SOD inhibitors or compounds used in the treatment of pathologies of the oral cavity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
