Comparison of native and recombinant chlorite dismutase from Ideonella dechloratans.
A detailed comparison between native chlorite dismutase from Ideonella dechloratans, and the recombinant version of the protein produced in Escherichia coli, suggests the presence of a covalent modification in the native enzyme. Although the native and recombinant N- and C-terminal sequences are identical, the enzymes display different electrophoretic mobilities, and produce different peptide maps upon digestion with trypsin and separation of fragments using capillary electrophoresis. Comparison of MALDI mass spectra of tryptic peptides from the native and recombinant enzymes suggests two locations for modification in the native protein. Mass spectrometric analysis of isolated peptides from a tryptic digest of the native enzyme identifies a possible cross-linked dipeptide, suggesting an intrachain cross-link in the parent protein. Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorbing at 295 nm, suggesting the presence of about one tyrosine residue per subunit with an anomalously low pK(a). The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination. These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found in other heme enzymes operating under highly oxidizing conditions.
Department of Chemistry, Karlstad University, Sweden.
Helena
H
Danielsson Thorell
Natascha H
NH
Beyer
Niels H H
NH
Heegaard
Marcus
M
Ohman
Thomas
T
Nilsson
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D010455:Peptides; D011994:Recombinant Proteins; D010088:Oxidoreductases; C402794:chlorite dismutase