title: Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium.
abstract: Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme from Bacillus megaterium that is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system. We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme. B. megaterium ATCC 14581 was grown to high cell density, and P450 102 was purified rapidly and in high yield by chromatography on adenosine-2',5'-diphosphate agarose from crude cell-free extract. The cytochrome bound to the column with remarkable avidity, in contrast to the significantly weaker binding observed for NADPH-cytochrome P450 reductase. Chromatographic behavior also showed that the cytochrome bound NADP(+)-type nucleotides more tightly than any other cellular polypeptide. The purified protein was electrophoretically homogeneous and had essentially theoretical contents of FAD, FMN, and heme. Optical spectra showed the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized. Molar extinction coefficients in the oxidized, fully reduced, and ferrous carbonyl states have been determined; notable is the large soret extinction in the ferrous carbonyl state (epsilon 449 nm = 143,500 M-1 cm-1). Final preparations were active in the oxidation of a wide variety of substrates. Of the C14 alkyl compounds studied, tetradecyltrimethylammonium bromide showed the highest substrate-dependent oxidation of NADPH, followed by myristate and myristyl alcohol; however, myristate exhibited the lowest Km value. Activities were tightly coupled to NADPH oxidation (> 97%). Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflourobutyrate, and 7-ethoxycoumarin were not substrates. NADP+ titrations showed, as expected, that the coenzyme was bound very tightly, with an average Kd of 580 nM. Our preparations of P450 102 are of sufficient purity and stability that crystals of the native holoenzyme have been grown.
authors.affiliation: Department of Biochemistry, University of Texas Health Center at Tyler 75710-2003.
authors.forename: S D
authors.initials: SD
authors.lastname: Black
authors.forename: M H
authors.initials: MH
authors.lastname: Linger
authors.forename: L C
authors.initials: LC
authors.lastname: Freck
authors.forename: S
authors.initials: S
authors.lastname: Kazemi
authors.forename: J A
authors.initials: JA
authors.lastname: Galbraith
mesh_terms: D001410:Bacillus megaterium / Q000187:drug effects / Q000201:enzymology*; D001426:Bacterial Proteins; D001463:Barbiturates / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001463:Barbiturates; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases