Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5

Truan, Gilles; Cullin, Christophe; Reisdorf, Philippe; Urban, Philippe; Pompon, Denis

doi:10.1016/0378-1119(93)90744-n

Cited by 149 publications

(110 citation statements)

References 25 publications

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…CYP2B6, 2C8, 2C9 and 3A4 were expressed in a previously described yeast strain W(R)fur1 [36] system, in which yeast cytochrome P450 reductase was overexpressed. Transformation by a pYeDP60 vector containing one of the human liver CYP2B6, 2C8, 2C9 and 3A4 cDNAs was then performed according to a general method of construction of yeast strain W(R)fur 1 expressing various human liver P450s [37][38][39].…”

Section: Origins Of Recombinant Cytochromes P450mentioning

confidence: 99%

“…The substrate concentrations used in these experiments were equal to the Km of the activity followed for each CYP (see Materials and Methods). Microsomes of the W(R)fur1 yeast strain expressing each CYP and overexpressing yeast cytochrome P450 reductase [36] were used. We have confirmed that the IC 50 values measured with these systems were very similar to those measured by using microsomes of insect cells expressing CYP 2B6, 2C8, 2C9, and 3A4 in the case of compounds 1, 3-6, 11, 13, 14 and 17 (less than 15 % variation of the IC 50 values).…”

Section: Selectivity Of the Inhibitors Towards Cyp2j2 By Comparison Wmentioning

confidence: 99%

See 1 more Smart Citation

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

Lafite

Dijols

Zeldin

et al. 2007

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

Twenty five derivatives of the drugs terfenadine and ebastine have been designed, synthesized, and evaluated as inhibitors of recombinant human CYP2J2. Compound 14, which has an imidazole substituent, is a good non competitive inhibitor of CYP2J2 (IC 50 = 400 nM). It is not selective towards CYP2J2 as it also efficiently inhibits the other main vascular CYPs, such as CYP2B6, 2C8, 2C9 and 3A4; however, it could be an interesting tool to inhibit all these vascular CYPs. Compounds 4, 5 and 13, which have a propyl, allyl and benzo-1,3-dioxole terminal groups, respectively, are selective CYP2J2 inhibitors. Compound 4 is a high-affinity, competitive inhibitor and alternative substrate of CYP2J2 (K i = 160 ± 50 nM). Compound 5 and 13 are efficient mechanism-based inhibitors of CYP2J2 (k inact /K I values ~3000 L.mol −1 .s −1 ). Inactivation of CYP2J2 by 13 is due to the formation of a stable iron-carbene bond which occurs upon CYP2J2-catalyzed oxidation of 13 with a partition ratio of 18 ± 3. These new selective inhibitors should be interesting tools to study the biological roles of CYP2J2.

show abstract

Section: Origins Of Recombinant Cytochromes P450mentioning

confidence: 99%

Section: Selectivity Of the Inhibitors Towards Cyp2j2 By Comparison Wmentioning

confidence: 99%

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

Lafite

Dijols

Zeldin

et al. 2007

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

show abstract

“…The ingredients for yeast culture media were purchased from Difco. Rabbit liver cytochrome b, was purified according to Strittmatter et al (1978) and was kindly provided by Dr D. Pompon (CGM, Gif-sur-Yvette, France) as well as the yeast strains W(R) engineered in their genomic DNA (Truan et al, 1993). Other chemicals and solvents used were of the highest quality commercially available.…”

Section: Experimental Procedures Chemicalsmentioning

confidence: 99%

“…In this study, we used yeast microsomes containing a human liver P450 3A4 [NF25-V8-W(R) or hPCN1-V8-W(R)] expressed in Saccharomyces cerevisiae as well as yeast control microsomes [VS-W(R)]. The W(R) strain, ( MAT a,le~2~,"',his3",",ura3',trpl',canR,cyr') which overexpresses yeast endogenous NADPH-P450 reductase (Truan et al, 1993) was used for expression.…”

Section: Yeast Strain Yeast Transformation Cell Culture and Microsomentioning

confidence: 99%

High affinity of ergopeptides for cytochromes P450 3A

Peyronneau

Delaforge

Riviere³

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy. Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone-treated rat microsomes. All the ergopeptides studied exhibited a high affinity for rat P450s 3A (K-around 1 pM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s. A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum. Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast. In both cases all the ergopeptides studied showed high affinities for the P450s (K? 0.6-2.2 pM) and an intense shift from the low-spin to the high-spin state upon substrate binding (60-100% spin shift). Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4.Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety. Human liver microsomes as well as P450 NF25 and P450 hPCNl also exhibited a high activity for bromocriptine hydroxylation at this level.These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily. The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type-I difference spectra. Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.Cytochrome P450 enzymes constitute a superfamily of heme-thiolate proteins that catalyse the primary oxidation of a wide variety of natural endogenous substrates like steroids, fatty acids, prostaglandins, leukotrienes and lipid hydroperoxides. They also play an important role in the metabolism of exogenous compounds like drugs, procarcinogens, solvents, anesthetics and environmental pollutants. Their broad substrate specificity is now well understood on the basis of enzyme multiplicity (Gonzalez, 1992). More than 220 P450s have been sequenced and characterized; they have been classified on the basis of primary amino acid sequence similarity (Nelson et...

show abstract

“…The strain W(R) was constructed from W(N) by the insertion of the GALlO-CYCl galactoseinducible promoter immediately upstream of the 5' end of the YRED open-reading frame (Truan et al, 1993). W(R)furl is a spontaneous and stable f u r l mutant of W(R) (Kern et al, 1990;Truan et al, 1993).…”

Section: Yeast Strainsmentioning

confidence: 99%

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

Bellamine

Gautier

Urban

et al. 1994

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

An expression library of hybrid cDNAs was constructed in vivo by homeologous recombination in yeast between human P450 1Al and P450 1 A2 sequences. Two clones exhibiting highly enhanced monooxygenase activities in vivo were selected. Chimera S12 includes the 88 N-terminal residues of P450 1Al fused to the complementary part of the P450 1A2 sequence. Chimera S71 derives from P450 1A1 by the substitution of the 36 C-terminal amino acid residues by the corresponding 38 residues of the 1A2 sequence. Biochemical analysis on microsomal fractions indicated that S12 and S71 have the same substrate specificities as 1A2 and 1A1, respectively. The observed increase in the in vivo monooxygenase activity is related to a ninefold increase in the microsomal S12 content as compared to the 1A2 content. In contrast, the expression level of S71 is slightly reduced but its turnover numbers are increased as compared to 1Al. The folding stability of chimeric P450 enzymes was evaluated by thermal and chaotropic agent denaturation. No difference was found between S12 and 1A2, but S71 appeared slightly less stable than 1Al. In vivo experiments indicated that S12 mRNA accumulation and stability are quite similar to the stability of parental 1A2 and, for both chimeras and parental enzymes, the protein half-lives are longer than the cell doubling time. The surprising accumulation of chimera S12 in the microsomal membrane is discussed in terms of the relationship of protein folding with transport to the endoplasmic reticulum membrane and the apparent expression levels of human P450 enzymes produced in yeast.

show abstract

Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5

Cited by 149 publications

References 25 publications

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

Selective, competitive and mechanism-based inhibitors of human cytochrome P450 2J2

High affinity of ergopeptides for cytochromes P450 3A

Chimeras of the Human Cytochrome P450 1A Family Produced in Yeast

Contact Info

Product

Resources

About