Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H.
Catalases are essential components of the cellular equipment to cope with oxidative stress. We have purified and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H. This oxidative stress-induced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid. We have used consecutive steps of hydrophobic, molecular sieve, and ion exchange chromatography to achieve a high state of purity for this metalloenzyme. The purified sample of catalase exhibited a specific catalytic activity of 55,900 U/mg, allosteric behavior in peroxidic reaction, a broad pH optimum, and a rather atypical electronic spectrum. The sample of highest purity was subjected to mass spectrometry analysis. The molecular weight of the subunit of this homodimeric protein was determined as 55,417 Da. The Qq-TOF mass analysis method allowed us to sequence short tryptic fragments of this catalase. Five such fragments with a total length of 57 amino acids together with several enzymatic properties allowed the classification of this hydroperoxidase as belonging to clade III of monofunctional catalases. The highest sequence similarity is with the catalase from Vibrio fischeri. The presented results imply the significance of this inducible enzyme in the prevention of toxic effects of oxidative stress for bacterial cells.
Zámocký
Marcel
M
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia. umikmzam@savba.sk
Godocíková
Jana
J
Gasperík
Juraj
J
Koller
Franz
F
Polek
Bystrík
B
D000595:Amino Acid Sequence; D002374:Catalase / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism; D020585:Comamonas / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014733:Vibrio / Q000235:genetics
D002374:Catalase