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-per oxidase (EC 1.11.1.7). We have established a system for the high level expr ession of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidi ne 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 chela te affinity and hydrophobic interaction chromatography. The homodimeric aci dic protein (pl = 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 ac tivity (apparent K-m = 4.9 +/- 0.25 mM and apparent k(cat) = 3500 s(-1)) an d an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrog allol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow sp ectroscopy, formation of compound I with peroxoacetic acid was calculated t o 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 re combinant enzyme indicated that both have the same heme environment. An app arent second-order rate constant for cyanide binding of (4.8 +/- 0.1) x 10( 5) M(-1)s(-1) was obtained.