Biochemical properties and regulated gene expression of the superoxide dismutase from the facultatively aerobic hyperthermophile Pyrobaculum calidifontis.
Superoxide dismutase (SOD) was purified from a facultatively aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis VA1. The purified native protein from aerobically grown cells exhibited 1,960 U of SOD activity/mg and contained 0.86 +/- 0.04 manganese and <0.01 iron atoms per subunit. The gene encoding SOD was cloned and expressed in Escherichia coli. Although the recombinant protein was soluble, little activity was observed due to the lack of metal incorporation. Reconstitution of the enzyme by heat treatment with either Mn or Fe yielded a highly active protein with specific activities of 1,970 and 434 U/mg, respectively. This indicated that the SOD from P. calidifontis was a cambialistic SOD with a preference toward Mn in terms of activity. Interestingly, reconstitution experiments in vitro indicated a higher tendency of the enzyme to incorporate Fe than Mn. When P. calidifontis was grown under anaerobic conditions, a majority of the native SOD was incorporated with Fe, indicating the cambialistic property of this enzyme in vivo. We further examined the expression levels of SOD and a previously characterized Mn catalase from this strain in the presence or absence of oxygen. Northern blot, Western blot, and activity measurement analyses revealed that both genes are expressed at much higher levels under aerobic conditions. We also detected a rapid response in the biosynthesis of these enzymes once the cells were exposed to oxygen.
Amo
Taku
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Atomi
Haruyuki
H
Imanaka
Tadayuki
T
D000332:Aerobiosis; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D007501:Iron; D008345:Manganese; D008969:Molecular Sequence Data; D041543:Pyrobaculum / Q000201:enzymology* / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D008345:Manganese; D007501:Iron; D002374:Catalase; D013482:Superoxide Dismutase