NADPH-cytochrome P-450 reductase of yeast microsomes

Aoyama, Yuri; Yoshida, Yasuhiko; Kubota, Sachiko; Kumaoka, Hiroshi; Furumichi, Atsuko

doi:10.1016/0003-9861(78)90178-9

Cited by 88 publications

(25 citation statements)

References 17 publications

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“…This observation might also be related with the presence of large amount of air-stable semiquinone in freshly purified SCR1 preparations. Nevertheless, semiquinone accumulation in the yeast reductase contrasted with the calculated redox potential inversion, rapid kinetic data and previous reports [46,63]. A possible explanation for the observed air-stable spectra would be stabilization of a FAD (and not FMN) semiquinone by NADP ϩ .…”

Section: Discussionsupporting

confidence: 46%

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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Section: Discussionsupporting

confidence: 46%

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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“…Purification of Soluble Yeast and Human CPR-Soluble yeast and human CPR purifications were carried out at 4°C and a flow rate of 1 ml/min as described previously, with few modifications, and omitting detergent in the purification steps (25). The cytosol of JL20 expressing soluble CPR was precipitated with ammonium sulfate, and the precipitate obtained with 40 -65% saturated ammonium sulfate was redissolved in 40 ml of Buffer B (10 mM potassium phosphate buffer, pH 7.0, containing 1 M FAD, 1 M FMN, 1 mM EDTA, and 10% (w/v) glycerol) and dialyzed overnight against 4 liters of 10 mM Buffer B.…”

Section: Methodsmentioning

confidence: 99%

The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

Kanamarlapudi¹,

Lamb²,

Kelly³

et al. 1998

Journal of Biological Chemistry

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The disruption of Saccharomyces cerevisiae NADPHcytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.

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“…Thus all subsequent experiments were carried out with 1 mM NADPH, a NADPH-regenating system under aerobic conditions and without CN -, unless otherwise indicated. Finally the reaction was not inhibited by menadione which is known to form an electron shunt from NADPH -cytochromc-P450 reductase to cytochrome P450 [26].…”

Section: Resultsmentioning

confidence: 96%

Microsomal Δ^8,14‐sterol Δ¹⁴‐reductase in higher plants

Taton¹,

Benveniste²,

Rahier³

1989

European Journal of Biochemistry

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An en7ymatic assay for d **'"-sterol A'"-reductase, an enzyme involved in sterol biosynlhcsis, has been developed for the first time in higher plants. The properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics and substrate specificity. This enzymatic double-bound reduction is thought to proceed through an electrophilic addition mechanism, involving a C14 putative carbonium ion highenergy intermediate. Using this in vitro assay, ammonium and iminium analogues of this cationic intermediate wcre shown to be potent inhibitors of the reduction reaction. Thus, compounds of the N-alkyl-8-aza-4a,lOdimethyl-trans-decal-3P-01 series strongly inhibited sterol reductase (I5" = 0.07 -4 pM) = 10-'), as did the antimycotic agent 15-azasterol (Iso = 0.03 pM); all of these compounds act as reaction-intermediate analogues of the proposed C14 carbonium ion intermediate. Moreover, the in vitro inhibition of the plant sterol rcductase by a series of ammonium-ion-containing fungicides was demonstrated. The relative specificity of these different series of inhibitors toward cycloeucalenol-obtusifoliol isomerase, 4 ' +d7-sterol isomerase and sterol d '"-reductase, was directly studied.The biosynthetic pathway of cholesterol in rat liver, and of ergosterol in yeast, includes a ~l~, '~-s t e r o l intermediate which has been recently shown to be the immediate metabolite of lanosterol demethylation at C32 by thc cytochrome-P450-dependent C14a demcthylase, both in rat livcr [l -31 and yeast [4, 51. The existence of an enzyme capable of reducing the ') double bond of S~-cholesta-8,14-dien-3P-o1 has been demonstrated in rat liver homogenate during cholesterol biosynthesis [6] and has been more recently characterized and partially purified [7]. Subsequently, A'-'4-sterol A' "-reductase activity was shown to exist in yeast microsomes, using ignosterol as substrate [8]. Although a ~l*"~)-sterol has been proposed as a possible intermediate in the biosynthesis of sterols in higher plants [9], the isolation of 5a-stigmasta-8,14,24 (28) PranceAbbreviations. GC/MS. coupled gas chromatographylmass spectroscopy; ZSO, the inhibitor concentration which rcduces the observed reaction rate by 50%; Rf, rctcntion factor: tR, relative retention time to cholesterol.Enqmes. ~i**'~-Sterol A14-reductasc (EC 1.3.1 .-); cycloeucalciiol-obtusifoliol isomerase (EC 5.5.1 9); A'+d'-sterol isomerasc (EC 5.3.3.5).Moreover, it has been shown in our laboratory that bramble cells cultured in vitro in the presence of the antimycotic agent 15-azasterol, a compound known to inhibit the reductasc in yeast [XI, accumulated large amounts of sterols [ 131, and more recently that 4a-methyl-5a-ergosta-8,14,24(28)-trien-3jl-ol (Fig. 1, compound 1) was the immediate demethylated metabolite of obtusifoliol (Fig. 1, compound 8) by the cytochrome-P450-dependent obtusifoliol 14a-demethylase isolated from Zea mays microsomes [14]. Since higher plants, when treated with AY9944 (Fig. 1, compound 20), an inhibitor of d8+d7-sterol isomerase...

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NADPH-cytochrome P-450 reductase of yeast microsomes

Cited by 88 publications

References 17 publications

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

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

The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

Microsomal Δ^8,14‐sterol Δ¹⁴‐reductase in higher plants

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NADPH-cytochrome P-450 reductase of yeast microsomes

Cited by 88 publications

References 17 publications

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

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

The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

Microsomal Δ8,14‐sterol Δ14‐reductase in higher plants

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Microsomal Δ^8,14‐sterol Δ¹⁴‐reductase in higher plants