title: Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4.
abstract: A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.
authors.forename: Boon Hooi
authors.identifier: 0000-0001-5252-0516
authors.initials: BH
authors.lastname: Tan
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia ; Institute of Biosciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.
authors.forename: Thean
authors.initials: T
authors.lastname: Chor Leow
authors.affiliation: Institute of Biosciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia ; Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.
authors.forename: Hooi Ling
authors.initials: HL
authors.lastname: Foo
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia ; Institute of Biosciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.
authors.forename: Raha
authors.initials: R
authors.lastname: Abdul Rahim
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007525:Isoelectric Focusing; D013294:Lactococcus lactis / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013482:Superoxide Dismutase