Catalase-Peroxidase from the Cyanobacterium Synechocystis PCC 6803: Cloning, Overexpression in Escherichia coli, and Kinetic Characterization
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Christa Jakopitsch
Abstract
The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pI = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent Km = 4.9 ± 0.25 mM and apparent kcat = 3 500 s−1) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 ± 0.26) × 103 M−1 s−1, whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 ± 0.03) × 106 M−1 s−1, (8.62 ± 0.21) × 104 M−1 s−1, and (5.43 ± 0.19) × 103 M−1 s−1, respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 ± 0.1) × 105 M−1 s−1 was obtained.
Copyright © 1999 by Walter de Gruyter GmbH & Co. KG
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- Execution of Apoptosis: Converging or Diverging Pathways?
- Two Highly Related Homeodomain Proteins, Nkx5-1 and Nkx5-2, Display Different DNA Binding Specificities
- The Janus Face of the Archaeal Cdc48/p97 Homologue VAT: Protein Folding versus Unfolding
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- Rat Muscle Fructose-1,6-Bisphosphatase: Cloning of the cDNA, Expression of the Recombinant Enzyme, and Expression Analysis in Different Tissues
- Catalase-Peroxidase from the Cyanobacterium Synechocystis PCC 6803: Cloning, Overexpression in Escherichia coli, and Kinetic Characterization
- Expression of Plasma Prekallikrein mRNA in Human Nonhepatic Tissues and Cell Lineages Suggests Special Local Functions of the Enzyme
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