Expression of Modified Human Cytochrome P450 1A1 in Escherichia coli: Effects of 5′ Substitution, Stabilization, Purification, Spectral Characterization, and Catalytic Properties

Guo, Z.; Gillam, Elizabeth M. J.; Ohmori, Shoko; Tukey, Robert H.; Guengerich, F. Peter

doi:10.1006/abbi.1994.1330

Cited by 105 publications

(57 citation statements)

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“…Since K m values varied little, the catalytic efficiency (V max /K m ) of the wild-type enzyme was likewise the highest with 7-ethoxyresorufin, whereas the analogous values for the other substrates were 10 times lower. It is worth mentioning that the apparent K m and V max values for 7-ethoxyresorufin O-deethyaltion observed in this study [i.e., 0.61 M and 11 nmol product formed min Ϫ1 (nmol P450) Ϫ1 , respectively, corresponded to those reported by Guo et al (1994) for their E. coli-expressed human P450 1A1, which were 0.58 M and 8.3 nmol product formed min…”

Section: Substrate Binding To Cytochrome P450 1a1supporting

confidence: 83%

“…The expression levels of all enzymes were fairly high, with that of the wild type close to 0.5 M, whereas the mutants were expressed at lower levels of 0.15 to 0.11 M. It should be noted that these values are derived from P450 measurements in sonicated spheroplasts. These levels are higher than those reported by Guo et al (1994) who first expressed recombinant human P450 1A1 in E. coli. As seen in Table 2, the total amount of purified P450 obtained from 1 liter culture was again the highest for the wild-type enzyme, ϳ 90 nmols, with close to a half of that amount in the case of the mutants.…”

Section: Molecular Modeling Analysescontrasting

confidence: 61%

“…Like mono-and bicistronic 1A1 clones, the His-tag-containing 1A1 wild-type and mutant enzymes were expressed in E. coli DH5␣ cells. The cultures were grown in 1 L of Terrific Broth medium containing 10 mg ampicillin liter Ϫ1 , 1 mM ␦-aminolevulinic acid (ALA), and 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for 48 h at 30°C with shaking at 120 to 150 rpm (Fisher et al, 1992;Guo et al, 1994). Membrane preparations were obtained essentially as described , by solubilization for 3 h at 4°C in 50 mM potassium phosphate buffer (pH 7.5) containing 20% glycerol (v/v), 1% Emulgen 911 and 1 mM PMSF.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Liu

Ericksen²,

Besspiata³

et al. 2003

Drug Metabolism and Disposition

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme. The Val-3823Ala mutation would increase the O-dealkylation of 7-pentoxyresorufin but decrease the oxidation of other substrates. In the case of the V382L mutant, the large bulk of Leu would block alkoxyresorufins from productive binding orientations leading to lowered activities. Binding free energy calculations for three substrates with 1A1 WT and two mutants indicated that binding constants would be similar for all enzyme-substrate combinations.Modeling predictions were tested experimentally. The plasmid containing the cDNA for human P450 1A1 modified for bacterial expression was altered to include a C-terminal PCR-generated six histidine domain to facilitate enzyme purification. The V382A and V382L mutants were constructed by site-directed mutagenesis and Escherichia coli-expressed enzymes purified using Ni-NTA affinity chromatography. The activity of the WT 1A1 was highest toward 7-ethoxyresorufin and lowest toward 7-pentoxyresorufin. Both mutants displayed a decrease in V max with 7-methoxy-and 7-ethoxyresorufin, whereas for the V382A mutant, V max with 7-pentoxyresorufin was increased. No significant changes in K m were observed relative to the wild-type enzyme. The experimental results are thus in good agreement with modeling predictions.

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Section: Substrate Binding To Cytochrome P450 1a1supporting

confidence: 83%

Section: Molecular Modeling Analysescontrasting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

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Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Liu

Ericksen²,

Besspiata³

et al. 2003

Drug Metabolism and Disposition

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“…purified CYP1A1, NADPH-cytochrome P450 reductase, which transfers electrons from NADPH to P450, and dilaurylglycerophosphocholine (Lau 2 PtdCho) [8][9][10]. However, this micellar system is not appropriate for studying the interactions between the components of the system as some of its properties are unlike those of the endoplasmic reticulum membrane, the natural environment of the microsomal monooxygenase system.…”

mentioning

confidence: 99%

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Kisselev¹,

Schwarz²,

Platt³

et al. 2002

Eur J Biochem

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Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo [a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmolAEmin )1 AEpmol )1 CYP1A1 of the enantiomeric promutagens (-)-and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both V max and K m values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1. ((-)-7,8-diol) and then metabolized by CYP1A1 to the ultimately genotoxic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (so-called diolepoxide-2 or antidiolepoxide, DE2) [1][2][3]. The last reaction appeared to be highly stereoselective as no or much less of the less carcinogenic r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydo-B[a]P (diolepoxide-1 or syn-diolepoxide, DE1) was produced from (-)-7,8-diol [4,5]. Racemic (+/-)-7,8-diol is mainly converted by human CYP1A1 to the DE2 [6,7].CYP1A1-dependent activity can be reconstituted by mixing the basic components of the monooxygenase system, i.e. purified CYP1A1, NADPH-cytochrome P450 reductase, which transfers electrons from NADPH to P450, and dilaurylglycerophosphocholine (Lau 2 PtdCho) [8][9][10]. However, this micellar system is not appropriate for studying the interactions between the components of the system as some of its properties are unlike those of the endoplasmic reticulum membrane, the natural environment of the microsomal monooxygenase system. This membrane is a bilayer, and it is highly probable that CYP1A1 and NADPH-cytochrome P450 reductase exhibit other important protein-lipid and protein-protein interactions there. Indeed, reconstitution systems using bilayer vesicles yielded higher rates of activity with rabbit liver CYP...

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“…The N-terminal modifications were as previously described [19,20]. Microsomes derived from AHH-1 TK+/-human lymphoblastoid B cell line co-expressed full-length cDNA of human CYPs: CYP2E1, CYP3A4, CYP1A2, CYP2C9 or CYP2D6 and the human cytochrome P450 reductase.…”

Section: Human Proteinsmentioning

confidence: 99%

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs

Lytton

Berg

Németh

et al. 2002

Clinical and Experimental Immunology

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SUMMARY Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti‐graft rejection and autoimmune disease therapy, is limited by their hepato‐ and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti‐CYP reactivity in 16% of children on CyA for anti‐graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8·5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti‐cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

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Expression of Modified Human Cytochrome P450 1A1 in Escherichia coli: Effects of 5′ Substitution, Stabilization, Purification, Spectral Characterization, and Catalytic Properties

Cited by 105 publications

References 0 publications

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane

Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs

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