Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.
Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.
Uhlmann
H
H
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Beckert
V
V
Schwarz
D
D
Bernhardt
R
R
D000325:Adrenodoxin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002417:Cattle; D003001:Cloning, Molecular; D003545:Cysteine; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D015870:Gene Expression; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis; D012694:Serine; D013329:Structure-Activity Relationship; D014162:Transfection
D008024:Ligands; D011994:Recombinant Proteins; D000325:Adrenodoxin; D012694:Serine; D003545:Cysteine