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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

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1437-4315
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Volume 380, Issue 9

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Catalase-Peroxidase from the Cyanobacterium Synechocystis PCC 6803: Cloning, Overexpression in Escherichia coli, and Kinetic Characterization

Christa Jakopitsch / Florian Rüker / Günther Regelsberger / Michael Dockal / Günter A. Peschek / Christian Obinger
Published Online: 2005-06-01 | DOI: https://doi.org/10.1515/BC.1999.135

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 K m = 4.9 ± 0.25 mM and apparent k cat = 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.

About the article

Published Online: 2005-06-01

Published in Print: 1999-09-13


Citation Information: Biological Chemistry, Volume 380, Issue 9, Pages 1087–1096, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.1999.135.

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