PubMed:10543446
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PubMed/sourceid/10543446","sourcedb":"PubMed","sourceid":"10543446","source_url":"http://www.ncbi.nlm.nih.gov/pubmed/10543446","text":"Catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803: cloning, overexpression in Escherichia coli, and kinetic characterization.\nThe 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.","tracks":[{"project":"CyanoBase","denotations":[{"id":"T1","span":{"begin":0,"end":19},"obj":"protein"},{"id":"T2","span":{"begin":170,"end":174},"obj":"protein"},{"id":"T3","span":{"begin":206,"end":225},"obj":"protein"},{"id":"T4","span":{"begin":621,"end":625},"obj":"protein"}],"attributes":[{"subj":"T1","pred":"source","obj":"CyanoBase"},{"subj":"T2","pred":"source","obj":"CyanoBase"},{"subj":"T3","pred":"source","obj":"CyanoBase"},{"subj":"T4","pred":"source","obj":"CyanoBase"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"CyanoBase","color":"#ec939d","default":true}]}]}}