Gene cloning and biochemical characterizations of thermostable ribonuclease HIII from Bacillus stearothermophilus.
The gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was 55 degrees C for Bst-RNase HIII and 35 degrees C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 degrees C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.
Chon
Hyongi
H
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 5665-0871, Japan.
Nakano
Rikita
R
Ohtani
Naoto
N
Haruki
Mitsuru
M
Takano
Kazufumi
K
Morikawa
Masaaki
M
Kanaya
Shigenori
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; C117079:ribonuclease HIII