Characterization of a hyperthermostable Fe-superoxide dismutase from hot spring.
A new gene encoding a thermostable Fe-superoxide dismutase (tcSOD) was identified from a metagenomic library prepared from a hot spring sample. The open reading frame of tcSOD encoded a 211 amino acid protein. The recombinant protein was overexpressed in Escherichia coli and confirmed to be a Fe-SOD with a specific activity of 1,890 U/mg using the pyrogallol method. The enzyme was highly stable at 80 degrees C and retained 50% activity after heat treatment at 95 degrees C for 2 h. It showed striking stability across a wide pH span from 4 to 11. The native form of the enzyme was determined as a homotetramer by analytical ultracentrifugation and gradient native polyacrylamide gel electrophoresis. Fe(2+) was found to be important to SOD activity and to the stability of tcSOD dimer. Comparative modeling analyses of tcSOD tetramer indicate that its high thermostability is mainly due to the presence of a large number of intersubunit ion pairs and hydrogen bonds and to a decrease in solvent accessible hydrophobic surfaces.
He
Yong-Zhi
YZ
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, P.O. Box 2714, Beijing, 100080, People's Republic of China.
Fan
Ke-Qiang
KQ
Jia
Cui-Juan
CJ
Wang
Zhi-Jun
ZJ
Pan
Wu-Bin
WB
Huang
Li
L
Yang
Ke-Qian
KQ
Dong
Zhi-Yang
ZY
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D002681:China; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015698:Genomic Library; D045482:Hot Springs; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D013482:Superoxide Dismutase