The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli.
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.
Mutsuda
M
M
Department of Food and Nutrition, Kinki University, Nara, Japan.
Ishikawa
T
T
Takeda
T
T
Shigeoka
S
S
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000201:enzymology*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria