Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability.
A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.
Huang
Jenq-Kuen
JK
Department of Chemistry, Western Illinois University, 1 University Circle, Macomb, Illinois 61455-1390, USA.
Wen
Lisa
L
Ma
Hsu
H
Huang
Zong-Xian
ZX
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D017377:Diatoms / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D013482:Superoxide Dismutase