Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.
A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000276:immunology; D001410:Bacillus megaterium / Q000201:enzymology*; D001463:Barbiturates / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D006900:Hydroxylation; D007700:Kinetics; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000032:analysis; D009249:NADP / Q000494:pharmacology; D010105:Oxygenases / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D000906:Antibodies; D001463:Barbiturates; D005227:Fatty Acids; D005415:Flavins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D009245:NADH Dehydrogenase