The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange.
An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced. The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively. To the best of our knowledge, this is the highest directly measured melting temperature of a native protein. Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites. Obviously it is not the incorporation of a metal ion that confers the extreme thermostability. Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.
Kardinahl
S
S
Institute of Biochemistry, Medical University of Lübeck, Germany.
Anemüller
S
S
Schäfer
G
G
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003300:Copper; D003460:Crystallization; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D019766:Genes, Archaeal; D007501:Iron; D008345:Manganese; D008670:Metals; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008670:Metals; D011993:Recombinant Fusion Proteins; D008345:Manganese; D003300:Copper; D007501:Iron; D013482:Superoxide Dismutase