Christa Jakopitsch, Florian Rüker, Günther Regelsberger, Michael Dockal, Günter A. Peschek, Christian Obinger
Catalase-Peroxidase from the Cyanobacterium
Synechocystis PCC 6803: Cloning, Overexpression in
Escherichia coli, and Kinetic Characterization
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.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 380, 09/1999
Pages: 1087 - 1096
Show full article (external site)
Show all available items of this journal
Search
