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Cloning, overproduction, and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus

(2001) EUROPEAN JOURNAL OF BIOCHEMISTRY. 268(24). p.6559-6568
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Abstract
The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c(2) and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (E-m=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c(2); the catalytic activities ('turnover number') are 85 800.min(-1) and 63 600.min(-1), respectively. These activities are the highest ever found for a BCCP.
Keywords
SPECIFICITY, OXIDATION, OXIDASE, PURIFICATION, EXPRESSION, NITRITE REDUCTION, PSEUDOMONAS-AERUGINOSA, ESCHERICHIA-COLI, PARACOCCUS-DENITRIFICANS, 2 INDEPENDENT PATHWAYS, peroxidase, overproduction, cytochrome, calcium, BCCP

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Citation

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Chicago
De Smet, Lina, Graham W Pettigrew, and Jozef Van Beeumen. 2001. “Cloning, Overproduction, and Characterization of Cytochrome c Peroxidase from the Purple Phototrophic Bacterium Rhodobacter Capsulatus.” European Journal of Biochemistry 268 (24): 6559–6568.
APA
De Smet, Lina, Pettigrew, G. W., & Van Beeumen, J. (2001). Cloning, overproduction, and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus. EUROPEAN JOURNAL OF BIOCHEMISTRY, 268(24), 6559–6568.
Vancouver
1.
De Smet L, Pettigrew GW, Van Beeumen J. Cloning, overproduction, and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus. EUROPEAN JOURNAL OF BIOCHEMISTRY. 2001;268(24):6559–68.
MLA
De Smet, Lina, Graham W Pettigrew, and Jozef Van Beeumen. “Cloning, Overproduction, and Characterization of Cytochrome c Peroxidase from the Purple Phototrophic Bacterium Rhodobacter Capsulatus.” EUROPEAN JOURNAL OF BIOCHEMISTRY 268.24 (2001): 6559–6568. Print.
@article{138969,
  abstract     = {The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c(2) and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (E-m=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c(2); the catalytic activities ('turnover number') are 85 800.min(-1) and 63 600.min(-1), respectively. These activities are the highest ever found for a BCCP.},
  author       = {De Smet, Lina and Pettigrew, Graham W and Van Beeumen, Jozef},
  issn         = {0014-2956},
  journal      = {EUROPEAN JOURNAL OF BIOCHEMISTRY},
  keyword      = {SPECIFICITY,OXIDATION,OXIDASE,PURIFICATION,EXPRESSION,NITRITE REDUCTION,PSEUDOMONAS-AERUGINOSA,ESCHERICHIA-COLI,PARACOCCUS-DENITRIFICANS,2 INDEPENDENT PATHWAYS,peroxidase,overproduction,cytochrome,calcium,BCCP},
  language     = {eng},
  number       = {24},
  pages        = {6559--6568},
  title        = {Cloning, overproduction, and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus},
  url          = {http://dx.doi.org/10.1046/j.0014-2956.2001.02610.x},
  volume       = {268},
  year         = {2001},
}

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