The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

Abstract

Synechococcus PCC 7942, a cyanobacterium, possesses catalaseperoxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2 +/- 0.27 mM and the kcat value was 2.6 x 10(4) s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3 +/- 0.84 and 20.2 +/- 0.95 microM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase-peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase-peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase-peroxidases of prokaryotic cells. Escherichia coli BL21 (DE3)plysS, harbouring a recombinant plasmid containing the catalase-peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

Increased cellular resistance to oxidative stress by expression of cyanobacterium catalase‐peroxidase in animal cells

“…2 shows a result of immunoblot analysis for the CPX expressed in the three transfectants. All of their extracts gave positive bands at a position of the subunit molecular mass (79 kDa) of S.7942 CPX [2], whereas the non-transfected cells and the cells transfected with pRc/CMV vector showed no immunoreactive band. The content of CPX protein expressed in the three transfectants varied in parallel with the level of their catalase activity.…”

“…Catalase activity was determined spectrophotometrically by measuring the decrease in absorbance at 240 nm in a 1.0 ml solution containing 50 mM sodium phosphate bu¡er (pH 7.0), 10.5 mM H P O P , and enzyme at 27³C [2]. One unit of enzyme was de¢ned as the quantity that catalyzes the decomposition of 1 Wmol of H P O P in 1 min.…”

“…A previously obtained clone encoding the S.7942 CPX gene (designated pSCP2) [2] was used as a template for PCR ampli¢cation. A sense primer (5P-AGACTGCCACCATGACAGCAACCCAGG-3P) corresponding to nucleotides 3^29 was synthesized such that the Kozak translational starting motif [4] was introduced around the initiation codon, and M13 reverse primer was used as an antisense primer.…”

“…Recently, we puri¢ed catalase-peroxidase (CPX) of the cyanobacterium Synechococcus PCC 7942 (S.7942) and also isolated its gene [2]. This enzyme is composed of two identical subunits with a molecular mass of 79 kDa and characterized by a low K m value for H P O P (4.6 mM).…”

Tansley Review No. 112

“…It is worth noting here that photosynthesis and the thiol-regulated enzymes might be less sensitive to oxidants in both unicellular algae and cyanobacteria, perhaps allowing these species to tolerate relatively high stromal H # O # concentrations (Takeda et al, 1995). Moreover, the recent cloning and purification of catalase from one cyanobacterium showed the enzyme to be very similar to catalases from non-photosynthetic bacteria, with higher peroxidatic activity and affinity for H # O # than plant catalases (Mutsada et al, 1996). These properties might have contributed to the protection against drought conferred on tobacco plants by overexpression of an E. coli catalase in the chloroplast (Shikanai et al, 1998).…”