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.