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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  (A 406 /A 280 ) 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) × 10 3  M −1  s −1 , whereas compound I reduction by  o -dianisidine, pyrogallol and ascorbate was determined to be (2.71 ± 0.03) × 10 6  M −1  s −1 , (8.62 ± 0.21) × 10 4  M −1  s −1 , and (5.43 ± 0.19) × 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) × 10 5  M −1  s −1  was obtained.

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