Cloning and expression of the catalase gene from the anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).
We identified a gene encoding a catalase from the anaerobic bacteria Desulfovibrio vulgaris (Miyazaki F), and the expression of its gene in Escherichia coli. The 3.3-kbp DNA fragment isolated from D. vulgaris (Miyazaki F) by double digestion with EcoRI and SalI was found to produce a protein that binds protoheme IX as a prosthetic group in E. coli. This DNA fragment contained a putative open reading frame (Kat) and one part of another open reading frame (ORF-1). The amino acid sequence of the amino terminus of the protein purified from the transformed cells was consistent with that deduced from the nucleotide sequence of Kat in the cloned fragment of D. vulgaris (Miyazaki F) DNA, which may include promoter and regulatory sequences. The nucleotide sequence of Kat indicates that the protein is composed of 479 amino acids per monomer. The recombinant catalase was found to be active in the decomposition of hydrogen peroxide, as are other catalases from aerobic organisms, but its K(m) value was much greater. The hydrogen peroxide stress against D. vulgaris (Miyazaki F) induced the activity for the decomposition of hydrogen peroxide somewhat, so the catalase gene may not work effectively in vivo.
Kitamura
M
M
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan. kitamura@bioa.eng.osaka-cu.ac.jp
Nakanishi
T
T
Kojima
S
S
Kumagai
I
I
Inoue
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002374:Catalase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D016969:Desulfovibrio vulgaris / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D018384:Oxidative Stress; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D020035:Holoenzymes; D011994:Recombinant Proteins; D006418:Heme; D006861:Hydrogen Peroxide; D002374:Catalase