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 (pl = 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 = 3500 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) x 10(3) M(-1) s(-1), whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 +/- 0.03) x 10(6) M(-1) S(-1), (8.62 +/- 0.21) x 10(4) M(-1) S(-1), and (5.43 +/- 0.19) x 10(3) 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) x 10(5) M(-1) S(-1) was obtained.
Jakopitsch
C
C
Arbeitsgruppe Biophysikalische Chemie, Institut für Physikalische Chemie der Universität Wien, Austria.
Rüker
F
F
Regelsberger
G
G
Dockal
M
M
Peschek
G A
GA
Obinger
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010544:Peroxidases; C076136:catalase HPI