Affinity Isolation and Characterization of Cytochrome-P450-102 (BM-3) from Barbiturate-Induced Bacillus megaterium

Black, Shaun D.; Linger, Marlin; Freck, L. C.; Kazemi, Sona; Galbraith, Jeanne

doi:10.1006/abbi.1994.1148

Cited by 32 publications

(31 citation statements)

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“…CYP6A1 was further purified by a combination of DEAE-Sepharose and octyl-Sepharose column chromatography to apparent electrophoretic homogeneity. P450 reductase was purified using phenylSepharose chromatography followed by affinity chromatography on 2Ј,5Ј-ADP-agarose (33). Detergents were removed on a DEAE-Sepharose column.…”

Section: Construction Of the Vector For Expression Of The House Fly Cmentioning

confidence: 99%

Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5

Guzov¹,

Houston

Murataliev³

et al. 1996

Journal of Biological Chemistry

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1 was first discovered in Cecropia silkworm larvae (1), but the functions of this membrane-bound heme protein have been most extensively studied in mammals (2, 3). A microsomal form of cyt b 5 is required for numerous biosynthetic and biotransformation reactions, which include P450-dependent reactions (3), desaturation of fatty acids (4), plasmalogen biosynthesis (5), and cholesterol biosynthesis (6, 7). A soluble form of cyt b 5 is involved in the reduction of methemoglobin in erythrocytes (8) and the biosynthesis of N-glyconeuraminic acid (9). A mitochondrial form, bound to the outer mitochondrial membrane, has been described in mammals as well (10). Cyt b 5 -like sequences are also found as part of larger polypeptides such as flavocytochrome b 2 , sulfite oxidase and nitrate reductase (11), probably as a result of gene fusion events. The role of cyt b 5 in microsomal P450-dependent monooxygenase reactions has been studied most extensively.P450s are a large superfamily of heme proteins which play a crucial role in the biosynthesis of a number of endogenous compounds (steroid hormones, vitamins D 3 , eicosanoids, and so forth) and in the activation or detoxification of a vast variety of xenobiotics. In many of these reactions, cyt b 5 is known to determine the fate of certain substrates by either stimulating (2, 3) or inhibiting (2, 12, 13) substrate metabolism, or even by influencing the type of reaction catalyzed (14). The stimulating effect of cyt b 5 has been thought to result from: 1) enhanced rate of the second electron transfer to P450 (12, 15, 16); 2) increased "coupling" of the reaction, i.e. inhibition of superoxide or hydrogen peroxide formation (2, 12, 17, 18); 3) allosteric effects (19,20); and 4) stimulation of the first electron transfer from P450 reductase to some P450s (21). However, the exact mechanism by which cyt b 5 affects P450-dependent reactions remains unclear.Insect P450s have been extensively studied because of their crucial role in the biosynthesis of hormones regulating insect growth, development, and reproduction (ecdysteroids and juvenile hormones) and in the biotransformation of foreign compounds of synthetic (insecticides) or natural (plant and microbial toxins) origin (22). Metabolism of insecticides by P450s is a major mechanism of insecticide resistance in insects (22, 23), and detoxification of plant toxins by P450s is thought to be an adaptation to the hazards of herbivory (24). Both CYP6A1, an insect P450, which is overproduced in insecticide-resistant strains of the house fly, and NADPH-dependent cytochrome P450 reductase, which provides electrons to P450s from NADPH, have been cloned from the house fly, Musca domestica (25, 26) and expressed in E. coli (27). We have found that epoxidation of the cyclodiene insecticide heptachlor by CYP6A1 is stimulated by rat microsomal cyt b 5 in a reconstituted system.2 Furthermore, immunological evidence for the involvement of cyt b 5 in several P450-dependent monooxygenase activities in house fly microsomes has been reported (28).Our...

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Section: Construction Of the Vector For Expression Of The House Fly Cmentioning

confidence: 99%

Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5

Guzov¹,

Houston

Murataliev³

et al. 1996

Journal of Biological Chemistry

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“…In mammalian CPRs, FMN hydroquinone is the electron donor to ferric P-450 and the air stable semiquinone state of FMN has the highest redox potential [36]. However in Bacillus megaterium P-450 BM3, which is a natural fusion-protein between a P-450 domain and a CPR domain [41,42], reduced FMN has a higher redox potential and FMN semiquinone acts as an electron source for P-450 reduction [43,44].These differences between mammalian and P-450 BM3 CPRs questioned if a similar situation could exist between other eukaryotic reductases for which limited biochemical characterizations were reported [11, 23, 45Ϫ51]. This study aimed to investigate differences existing in thebinding, redox potentials and electron transfer properties of recombinant yeast, plant and mammalian CPRs and to determine consequences on their functions.…”

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confidence: 99%

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confidence: 99%

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

Louérat-Oriou

Perret

Pompon

1998

European Journal of Biochemistry

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Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/ reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/ semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest k cat whereas the yeast reductase gave the best k cat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activies with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.Keywords : NADPH-cytochrome P-450 reductase; redox potential ; kinetic property; P-450 interaction.NADPH-P-450 reductase (CPR; NADPH : ferrihemoprotein reductase) is a membrane-anchored protein that catalyzes electron transfers from NADPH to cytochrome P-450 (P-450) [1Ϫ4], heme oxygenase, cytochrome b 5 and squalene epoxidase [5,6]. The various members of the P-450 superfamily [7] are involved in CPR-supported-oxidative metabolism of a wide range of xenobiotics and endogenous compounds [8]. Contrasting with the large variety of P-450s, a single CPR is generally present in each organism [9,10]. Unlike animals and yeast, higher plants express multiple forms of CPRs as inferred from western blot analysis of purified materials [11,12]. Durst et al.[13] have isolated two distinct partial length cDNA from Helianthus tuberosus, corresponding to distinct mRNA species. The two CPRs isolated from parsley were 80% identical in amino acid sequence and a distinct metabolic role was clearly assigned to each other [14]. In Arabidopsis thaliana, two distantly related Enzymes. NADPH-cytochrome P-450 reductase (EC 1.6.2.4); cytochrome P-450 (EC 1.14.14.1).reductases (ATR1 and ATR2, 64% amino acid sequence identity) were cloned and may ...

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“…Alternatively, affinity chromatography on 2'5'-ADP-Sepharose can be used which has proven most successful with fusion proteins such as P450 BM-3 (Black et al 1994). This method is very efficient though expensive.…”

Section: Expensive Isopropyl-beta-d-thiogalactopyranoside (Iptg) Is Omentioning

confidence: 99%

Microbial P450 enzymes in biotechnology

Urlacher

Lutz-Wahl

Schmid

2004

Applied Microbiology and Biotechnology

200

100

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Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.3

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Affinity Isolation and Characterization of Cytochrome-P450-102 (BM-3) from Barbiturate-Induced Bacillus megaterium

Cited by 32 publications

References 0 publications

Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5

Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5

Differential redox and electron‐transfer properties of purified yeast, plant and human NADPH‐cytochrome P‐450 reductases highly modulate cytochrome P‐450 activities

Microbial P450 enzymes in biotechnology

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