Cloning and expression of the superoxide dismutase gene from the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).
We identified the SOD gene in the obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F) and constructed a high-level expression system in Escherichia coli. A 2.6-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SmaI contained the SOD gene and part of another open reading frame. The amino acid sequence deduced from the SOD gene, which was composed of 238 amino acid residues, showed high homogeneity with iron-containing SOD (Fe-SOD) and predicted that the amino terminus of this protein would carry an export signal peptide. We produced the precursor form of SOD (PSOD) and the mature form of SOD (MSOD), which lacked the putative signal peptide. In E. coli, PSOD was present in insoluble inclusion bodies, and its putative signal peptide was not cleaved. In contrast, MSOD contained one iron per mononer and formed a dimer, which exhibited an SOD activity of 850 U/mg. Furthermore, D. vulgaris soluble extract showed a band of SOD activity on native polyacrylamide gel that migrated to the same point as MSOD. The intracellular localization of SOD and its role in D. vulgaris are also discussed.
Nakanishi
Takeshi
T
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan.
Inoue
Hideo
H
Kitamura
Masaya
M
D000595:Amino Acid Sequence; D000693:Anaerobiosis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D016969:Desulfovibrio vulgaris / Q000201:enzymology* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D013482:Superoxide Dismutase