Characterization of Fe/Mn-superoxide dismutase from diatom Thallassiosira weissflogii: cloning, expression, and property.
A cDNA clone of 1114 bp encoding a putative Mn-superoxide dismutase (Mn-SOD) from diatom Thallassiosira weissflogii was cloned by the PCR technique. Nucleotide sequence analysis of this cDNA clone revealed that it was translated into 201 amino acid residues. When the sequence was compared with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed higher homology to Mn-SOD. The amino acid residues required to coordinate the single manganese ion were conserved in all reported Mn-SOD sequences. This cDNA was introduced in an expression vector, pET-20b(+), and transformed into E. coli BL21(DE3)pLysS. The expressed SOD protein was then purified by a His-tag column. The recombinant enzyme was heated at 55 degrees C with a time-dependent assay; the time interval for 50% inactivation was 23 min, and its thermal inactivation rate constant K(d) was 3.03 x 10(-)(2) min(-)(1). The enzyme was inactivated either in acidic pH (below 4.0) or in the presence of imidazole (above 1.6 M) and had only a moderate effect under SDS (above 4%), whereas it was not affected under an alkaline pH (above 9.0). The atomic absorption spectrometric assay showed that 0.6 atom of iron/manganese (3:1) was present in each subunit of SOD. Reconstitution study was suggested that diatom SOD was cambialistic (Fe/Mn)-SOD. The finding of this SOD cDNA could be used for a reference in comparing the differences among marine phytoplankton species and as a probe to detect the transcription level of this enzyme, which can be applied in cosmetics for skin protection or defending unesthetic effects caused by oxygen-containing free radicals.
Ken
Chuian-Fu
CF
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, Taiwan 202, Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan.
Hsiung
Tung-Ming
TM
Huang
Zong-Xian
ZX
Juang
Rong-Huay
RH
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017377:Diatoms / Q000201:enzymology*; D015870:Gene Expression; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008345:Manganese / Q000032:analysis; D008969:Molecular Sequence Data; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D008345:Manganese; D007501:Iron; D013482:Superoxide Dismutase