[6] Yeast expression of animal and plant P450s in optimized redox environments

Pompon, Denis; Louerat, Benedicte; Bronine, Alexis; Urban, Philippe

doi:10.1016/s0076-6879(96)72008-6

Cited by 601 publications

(687 citation statements)

References 21 publications

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“…The amplicons were cloned into p-GEMTeasy vector and sequenced using ABI310 capillary sequencer. After BglII and EcoRI digestion, the fragments were directionally cloned into the expression cassette of pYeDP60 (Pompon et al 1996) digested with BamHI and EcoRI. Saccharomyces cerevisiae strain WAT11 is a derivative from strain W303-1B, in which yeast CPR gene (coding for a NADPH-cytochrome P450 reductase) was replaced by the Arabidopsis ATR1 (Urban et al 1997).…”

Section: Methodsmentioning

confidence: 99%

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Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

et al. 2009

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Globe artichoke represents a natural source of phenolic compounds with dicaffeoylquinic acids along with their biosynthetic precursor chlorogenic acid (5-caffeoylquinic acid) as the predominant molecules. We report the isolation and characterization of a full-length cDNA and promoter of a globe artichoke p-coumaroyl ester 3 0 -hydroxylase (CYP98A49), which is involved in both chlorogenic acid and lignin biosynthesis. Phylogenetic analyses demonstrated that this gene belongs to the CYP98 family. CYP98A49 was also heterologously expressed in yeast, in order to perform an enzymatic assay with pcoumaroylshikimate and p-coumaroylquinate as substrates. Real Time quantitative PCR analysis revealed that CYP98A49 expression is induced upon exposure to UV-C radiation. A single nucleotide polymorphism in the CYP98A49 gene sequence of two globe artichoke varieties used for genetic mapping allowed the localization of this gene to linkage group 10 within the previously developed maps.

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Section: Methodsmentioning

confidence: 99%

“…Enzymatic assay of the C3 0 H activities Yeast microsomes were isolated after 24 h of induction on 20 g/l galactose at 30°C, according to Pompon et al (1996). The amount of total protein in microsomes was determined according to Bradford protein assay.…”

Section: Methodsmentioning

confidence: 99%

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

et al. 2009

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“…The full length gene was re-cloned, using the NotI/PacI restriction sites, into the yeast expression vector pYEDP60 [17] which was modified to contain PacI and NotI sites at the polylinker. The candidate chicory P450 was co-transformed with either a germacrene A synthase [18], amorpha-4,11-diene synthase [14] or a valencene synthase [19] into the into yeast strain WAT 11 expressing Arabidopsis ATR1 NADPH-cytochrome P450 reductase [20].…”

Section: Co-expression Of Cyp71av8 With Terpene Synthases In Yeastmentioning

confidence: 99%

“…The candidate chicory P450 was co-transformed with either a germacrene A synthase [18], amorpha-4,11-diene synthase [14] or a valencene synthase [19] into the into yeast strain WAT 11 expressing Arabidopsis ATR1 NADPH-cytochrome P450 reductase [20]. Germacrene A synthase was cloned into pYEDP80 vector [17] with TRP1 auxotrophic selection marker using BamHI and EcoRI restriction sites. Amorpha-4,11-diene synthase and valencene synthase were both cloned into pYES3/CT yeast expression vector (Invitrogen) with TRP1 selection marker using BamHI and NotI restriction sites.…”

Section: Co-expression Of Cyp71av8 With Terpene Synthases In Yeastmentioning

confidence: 99%

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

et al. 2010

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a b s t r a c tChicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.

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“…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]. Yeast culture and microsomes preparation were performed by using previously described techniques [40]. Microsomes were homogenized in 50 mM Tris buffer (pH 7.4) containing 1 mM EDTA and 20% glycerol (v/v), aliquoted, frozen under liquid N 2 , and stored at −80 °C until use.…”

Section: Origins Of Recombinant Cytochromes P450mentioning

confidence: 99%

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

Lafite

Dijols

Zeldin

et al. 2007

Archives of Biochemistry and Biophysics

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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.

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[6] Yeast expression of animal and plant P450s in optimized redox environments

Cited by 601 publications

References 21 publications

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

Isolation and mapping of a C3′H gene (CYP98A49) from globe artichoke, and its expression upon UV-C stress

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

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

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