Rubrerythrin from Clostridium perfringens: cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function.
The food-borne pathogen Clostridium perfringens, which is an obligate anaerobe, showed growth under conditions of oxidative stress. In protein extracts we looked for superoxide dismutase (SOD) activities which might scavenge highly toxic superoxide radicals evolving under such stress conditions. Using the classical assay to detect SOD activity on gels after electrophoresis of C. perfringens proteins, we obtained a pattern of three major bands indicating SOD activity. The protein representing the brightest band was purified by three chromatographic steps. On the basis of 20 amino acids determined from the N terminus of the protein, we designed a degenerate oligonucleotide probe to isolate the corresponding gene. We finally sequenced an open reading frame of 195 amino acids (molecular mass, 21,159 Da) with a strong homology to the Desulfovibrio vulgaris rubrerythrin; therefore, we assumed to have cloned a rubrerythrin gene from C. perfringens, and we named it rbr. The C-terminal region of the newly detected rubrerythrin from C. perfringens contains a characteristic non-heme, non-sulfur iron-binding site -Cys-X-X-Cys-(X)12-Cys-X-X-Cys- similar to that found in rubrerythrin from D. vulgaris. In addition, three -Glu-X-X-His- sequences could represent diiron binding domains. We observed SOD activity in extracts of Escherichia coli strains containing the recombinant rbr gene from C. perfringens. A biological function of rubrerythrin as SOD was confirmed with the functional complementation by the rbr gene of an E. coli mutant strain lacking SOD activity. We therefore suppose that rubrerythrin plays a role as a scavenger of oxygen radicals.
Lehmann
Y
Y
Laboratorium für Lebensmittel-Mikrobiologie, Institut für Lebensmittelwissenschaften, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Meile
L
L
Teuber
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology*; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D006422:Hemerythrin; D008969:Molecular Sequence Data; D012416:Rubredoxins; D013482:Superoxide Dismutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005288:Ferredoxins; D006422:Hemerythrin; D012416:Rubredoxins; C055857:rubrerythrins; D013482:Superoxide Dismutase