Unusual stability of manganese superoxide dismutase from a new species, Tatumella ptyseos ct: its gene structure, expression, and enzyme properties.
A genomic DNA of 1416 bp containing an open reading frame encoding a manganese superoxide dismutase (Mn-SOD) from Tatumella ptyseos ct was cloned. Sequence analysis of this new gene revealed that it translates 205 amino acid residues. The deduced amino acid sequence showed variable identities (41-91%) with sequences of Mn-SODs from other species. The residues required to coordinate the single trivalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are in other reported Mn-SODs. In addition, the gene was introduced into the expression vector, pET-20b(+), and transformed in Escherichia coli BL21(DE3). The Mn-SOD was purified by a His-tag technique. The yield was 0.9 mg from 0.5 L of culture. The specific activity was 6540 U/mg. A dimer is the major form of the enzyme in equilibrium. The half-life of dimer is approximately 50 min and its thermal inactivation rate constant k(d) was 0.015 min(-1) at 80 degrees C. The dimerization of the enzyme was inhibited under an acidic pH (below 4.0), or in the presence of SDS (above 1%) or imidazole (above 0.5 M), whereas it was not affected under an alkaline pH (above 9.0). Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin and chymotrypsin. This unusually stable enzyme can be used as cosmetic to the protection of skin against the unaesthetic effects caused by free radicals.
Ken
Chuian-Fu
CF
Institute of Biotechnology, National Changhua University of Education, Changhua 500, Taiwan.
Lee
Chuing-Chi
CC
Duan
Kow-Jen
KJ
Lin
Chi-Tsai
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000201:enzymology*; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D013482:Superoxide Dismutase