Molecular cloning and characterization of Mn-superoxide dismutase from disk abalone (Haliotis discus discus).
The mitochondrial enzyme manganese superoxide dismutase (mitMn-SOD) is one of the antioxidant enzymes involved in cellular defense against oxidative stress and catalyzes the conversion of O(2)(-) into the stabler H(2)O(2). In this study, a putative gene encoding Mn-SOD from disk abalone (Haliotis discus discus, aMn-SOD) was cloned, sequenced, expressed in Escherichia coli K12(TB1) and the protein was purified using pMAL protein purification system. Sequencing resulted ORF of 681 bp, which corresponded to 226 amino acids. The protein was expressed in soluble form with molecular weight of 68 kDa including maltose binding protein and pI value of 6.5. The fusion protein had 2781 U/mg activity. The optimum temperature of the enzyme was 37 degrees C and it was active in a range of acidic pH (from 3.5 to 6.5). The enzyme activity was reduced to 50% at 50 degrees C and completely heat inactivated at 80 degrees C. The alignment of aMn-SOD amino acid sequence with Mn-SODs available in NCBI revealed that the enzyme is conserved among animals with higher than 30% identity. In comparison with human mitMn-SOD, all manganese-binding sites are also conserved in aMn-SOD (H28, H100, D185 and H189). aMn-SOD amino acid sequence was closer to that of Biomphalaria glabrata in phylogenetic analysis.
Ekanayake
Prashani Mudika
PM
Department of Marine Biotechnology, Cheju National University, 66 Jejudaehakno, Jeju 690-756, Republic of Korea.
Kang
Hyun-Sil
HS
De Zyosa
Mahanama
M
Jee
Youngheun
Y
Lee
Youn-Ho
YH
Lee
Jehee
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D049851:Gastropoda / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D013696:Temperature
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013482:Superoxide Dismutase