Superoxide Reductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: its Function, Regulation, and Biotechnological Applications

Abstract

The anaerobic hyperthermophilic archaeon, Pyrococcus furious, possesses a system for the detoxification of reactive oxygen species, which is different from the classical defense mechanisms present in aerobes. P. furiosus employs a novel enzyme system centered on the enzyme superoxide reductase (SOR), which reduces superoxide molecules to hydrogen peroxide without producing oxygen. Surprisingly, P. furiosus SOR, unlike many P. furiosus enzymes, was shown to function at low temperature (<25o C). A model for superoxide reduction by SOR was proposed where the electrons used by SOR to reduce superoxide are supplied by a small iron containing protein, rubredoxin (Rd), and Rd is reduced by the oxidoreductase, NAD(P)H-rubredoxin oxidoreductase (NROR). The first objective of this study was to evaluate the validity of the proposed superoxide reduction pathway by using the recombinant SOR, Rd and NROR enzymes in an in vitro assay as well as to demonstrate in vivo function via complementation studies in superoxide detoxification deficient Escherichia coli strains. The second objective was to investigate the transcriptional expression levels of genes that are involved in the SOR-centered superoxide reduction pathway in order to determine how these genes are expressed and regulated in response to various oxidative stresses. The third objective was to evaluate the efficacy of the biotechnological application of this superoxide detoxification system by expressing SOR in plant cells, which enhanced their survival at high temperature and from drought indicating that it functions successfully in vivo. The fourth objective of this study was the characterization of glutathione reductase (GR) from a psychrophile, Colwellia psychrerythraea, which is stable at low temperatures and protects cells from free radicals by serving as a reductant. The C. psychrerythraea GR gene was cloned into an E. coli-based recombinant expression system. Recombinant C. psychrerythrae GR was expressed and purified. The recombinant GR showed significant activity at low temperature (4C). The P. furiosus superoxide reduction system genes and GR from C. psychrerythraea can be engineered into plants (Arabidopsis) to aid in combating damage caused by oxidative stress when plants undergo rapid changes in temperature, high light or UV exposure, or drought conditions.