Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269.
A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.
Seo
Sung Nam
SN
Molecular Microbiology Laboratory, Department of Biology, Yonsei University, Seoul 120-749, Korea.
Lee
Jae Ho
JH
Kim
Young Min
YM
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D018604:Epitope Mapping; D007501:Iron / Q000378:metabolism; D007526:Isoelectric Point; D008345:Manganese / Q000378:metabolism; D020631:Methylobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase