Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase 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 2.6×104 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 μM 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.
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May 15 1996
The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli Available to Purchase
Michinori MUTSUDA;
Michinori MUTSUDA
1Department of Food and Nutrition, Kinki University, Nakamachi, Nara 631, Japan
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Takahiro ISHIKAWA;
Takahiro ISHIKAWA
1Department of Food and Nutrition, Kinki University, Nakamachi, Nara 631, Japan
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Toru TAKEDA;
Toru TAKEDA
1Department of Food and Nutrition, Kinki University, Nakamachi, Nara 631, Japan
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Shigeru SHIGEOKA
Shigeru SHIGEOKA
*
1Department of Food and Nutrition, Kinki University, Nakamachi, Nara 631, Japan
*To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
November 06 1995
Revision Received:
January 15 1996
Accepted:
January 24 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 316 (1): 251–257.
Article history
Received:
November 06 1995
Revision Received:
January 15 1996
Accepted:
January 24 1996
Citation
Michinori MUTSUDA, Takahiro ISHIKAWA, Toru TAKEDA, Shigeru SHIGEOKA; The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli. Biochem J 15 May 1996; 316 (1): 251–257. doi: https://doi.org/10.1042/bj3160251
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