Substrate binding to 15beta-hydroxylase (CYP106A2) probed by FT infrared spectroscopic studies of the iron ligand CO stretch vibration.
CYP106A2 has been expressed in E. coli with a high yield of up to 130 mg per litre of culture, purified to electrophoretic homogenity and found to be active in 15beta-hydroxylation of deoxycorticosterone using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. Inspite of catalytic activity no substrate binding was detectable by UV-Vis spectroscopy. In contrast, an effect of substrate binding has been detected using the CO stretch mode infrared spectrum indicating that deoxycorticosterone binds in the heme pocket near the iron ligand.
Simgen
B
B
Universität des Saarlandes, Fachrichtung 12.4-Biochemie, Saarbrücken, D-66041, Germany.
Contzen
J
J
Schwarzer
R
R
Bernhardt
R
R
Jung
C
C
D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System