Multiple posttranslational modifications at distinct sites contribute to heterogeneity of the lipoprotein cytochrome bo(3).
The heme-copper cytochrome oxidase of Escherichia coli (cytochrome bo(3)) was tagged with oligohistidine at the C-terminus of the small noncatalytic subunit IV. After detergent solubilization, the enzyme was purified by a one-step procedure with immobilized metal affinity chromatography. Using different cytochrome bo(3) constructs as reference, the products were investigated by mass spectroscopical and immunological methods. Several posttranslational modifications of subunits II, III, and IV were observed: (1) N-terminal methionines of subunits III and IV are split off. (2) Fifty percent of subunit III polypeptides are acetylated, presumably at the N-terminal alanine. (3) Lipoprotein processing of subunit II involves cleavage of the signal peptide. (4) Maturation of subunit II [Ma, J., Katsonouri, A., and Gennis, R. B. (1997) Biochemistry 36, 11298-11303] alters the structure of the N-terminal cysteine by N-palmitoylation and S-glyceryldipalmitoylation. (5) A hexapeptide is split off from the C-terminus of subunit II. This happens subsequently to the N-terminal lipoprotein processing step and is dependent on the growth state of cells.
Prutsch
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Lehrstuhl Biophysik and Institut für Physiologische Chemie, Ruhr-Universität Bochum, Universitätsstrasse 150, D-44780 Bochum, Germany.
Lohaus
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Green
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Meyer
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Lübben
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018629:Spectrometry, Mass, Secondary Ion; D017550:Spectroscopy, Fourier Transform Infrared
D003573:Cytochrome b Group; D003580:Cytochromes; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C083850:cytochrome bo3, E coli