Isolation and characterization of cytochrome P-450meg.
The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.
D001410:Bacillus megaterium / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification*; D007700:Kinetics; D008667:Metalloproteins / Q000302:isolation & purification*; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013379:Substrate Specificity
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases