Activities and Kinetic Mechanisms of Native and Soluble NADPH–Cytochrome P450 Reductase

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  • Activ atNAD

    David udDianeInstitut ery

    Received

    Nativtase (C33 aminpressedThe prN-termstep usSodiumphoresthe profunctioductionD22-desc redunism wto prodzyme. TferenceafterdesatuNH2-tedispen

    Key Wtase; cyaction

    NADPHcytochrome P450 oxidoreductase (CPR; EC1.6.2.4) is a membrane-bound flavoprotein involvedwith cysubstrathe CYstratesadditioinstancthesis (

    lecascyaasqus is

    s t51as51steeo

    r e, C

    merres,ngeththeemfolth

    ctaance of fungus to azole drugs.

    1 Both2 Prese

    Bristol, B3 To w

    622350.

    Biochemical and Biophysical Research Communications 286, 4854 (2001)

    doi:10.1006/bbrc.2001.5338, available online at http://www.idealibrary.com on

    0006-291Copyright All rights otochrome P450 (CYP) in hydroxylation of itstes by transferring electrons from NADPH toP molecule (1, 2). In fungi, individual CYP sub-can be pollutants (for review, 3) or steroids (4);nally, CYPs can be biosynthetic enzymes of, fore, terpene, alkaloid, aflatoxin or sterol biosyn-for review, 5). CPR also has the ability to trans-

    Here the proteins were produced as N-terminallyappended his-tagged derivatives following heterolo-gous expression in Escherichia coli, purified to homo-geneity and demonstrated both to be catalytically ac-tive in cytochrome c reduction and reconstitution ofergosterol biosynthesis in cell lysates of a CPR dis-rupted yeast strain. Understanding the structure/function/mechanism of this enzyme at the molecularlevel will help in the development of antifungal drugsblocking essential electrons transfer during ergosterol

    authors contributed equally to this work.nt address: Department of Pharmacology, University ofristol, BS8 1TD, UK.ities and Kinetic Mechanisms of NPHCytochrome P450 Reductase

    C. Lamb,1 Andrew G. S. Warrilow,1 KanamarlapE. Kelly, and Steven L. Kelly3

    e of Biological Sciences, University of Wales Aberystwyth, Ab

    June 25, 2001

    e yeast NADPHcytochrome P450 oxidoreduc-PR; EC 1.6.2.4) and a soluble derivative lackingo acids of the NH2-terminus have been overex-as recombinant proteins in Escherichia coli.

    esence of a hexahistidine sequence at theinus allowed protein purification in a singleing nickel-chelating affinity chromatography.

    dodecyl sulfatepolyacrylamide gel electro-is confirmed the predicted molecular weights ofteins and indicated a purity of >95%. Proteinnality was demonstrated by cytochrome c re-

    and reconstitution of CYP61-mediated sterolaturation. Steady-state kinetics of cytochromectase activity revealed a random Bi-Bi mecha-ith NADPH donating electrons directly to CPRuce a reduced intermediary form of the en-he kinetic mechanism studies showed no dif-between the two yeast CPRs in mechanism orreconstitution with CYP61-mediated 22-

    ration, confirming that the retention of therminable membrane anchor is functionallysable. 2001 Academic Press

    ords: NADPHcytochrome P450 oxidoreduc-tochrome P450; purification; reconstitution; re-mechanism.

    fer eygenferrisuchparapatemycetron(CYP

    YeCYPlanois thcommotheCPRCYPformwheimalducinot lingsyst200-thanredubiosyntCYP m

    hom correspondence should be addressed. Fax: (0044) 1970E-mail: Steven.Kelly@aber.ac.uk.

    48X/01 $35.002001 by Academic Press

    f reproduction in any form reserved.ive and Soluble

    i Venkateswarlu,2

    stwyth SY23 3DA, United Kingdom

    trons to several other heme proteins (heme ox-e, cytochrome c and cytochrome b5), potassiumnide, therapeutically important compoundsanticancer drug mitomycin c and the herbicide

    at (for review, 6). As noted above CPR partici-n sterol biosynthesis and in the yeast Saccharo-cerevisiae is defined as Ncrp1p donating elec-o squalene epoxidase, sterol 14a-demethylase) and sterol D22-desaturase (CYP61) (7).

    t cytochrome P45014dm (sterol 14a-demethylase;) catalyzes the removal 14a-methyl group fromrol in the ergosterol biosynthetic pathway andtarget for azole antifungal drugs, which arenly used in agriculture and medicine (8). Twonzymes of yeast ergosterol biosynthesis requireYP61 a sterol 22-desaturase (911) and a non-onooxygenase, squalene epoxidase (12). Themay also be present in other Kingdoms of Life

    sterol 22-desaturation is observed unlike in an-but the latter is present in all organisms pro-sterol (13). A yeast NCPR1 gene disruption isal, unlike CYP51 gene disruption (14), indicat-presence of an alternative electron donating

    (7). However NCPR1 gene disrupted strains ared more sensitive to ketoconazole (azole drug)e non-disrupted strain (15). This indicates these may have an indirect involvement in toler-hesis, to understand the mechanism of fungalediated xenobiotic metabolism and to unravel

  • the mechanisms for reductive metabolism of pesticidesand pollutants.

    MATER

    ChemiCompanpreparattific (LouFine Che

    CloninCPR:pETkb SalIYEp51 (HindIIIlinker. Sby subclgene, relBamHI sDNA maas descri

    Overexin E. coprocedurtransformwere grobroth (LBmg/ml). Hisopropywith shaoptical dby centrwas resucontaininlysate wdebris anfractionsbrane frawas deteusing bo

    Purificsoluble Caffinitywith 2%solubilizCPR wecolumn (buffer (2mM glycbuffer anmM imidlinear grand elutpurifiedtion assasium pho30 kDa msteps wewas perfstandardb-galactoalbuminkDa). ThR-250 to

    Reconstitution of Sterol D22-desaturase activity with native andsoluble CPR. Yeast cytochrome sterol D22-desaturase (CYP61) waspurified from yeast microsomal fractions as described previously

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    Vol. 286, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSIALS AND METHODS

    cals. All chemicals were obtained from Sigma Chemicaly (Poole, UK), unless otherwise stated. Chemicals for theion of phosphate buffers were obtained from Fisher Scien-ghborough, UK). Sodium cholate was obtained from Acrosmicals (Loughborough, UK).

    g of native and soluble yeast CPRs for expression in E. coli.15b expression vector was constructed by subcloning a 2.1

    HindIII fragment containing CPR gene, released from CPR:16), into XhoIBamHI site of pET15b using a 0.35 kbBamHI fragment, obtained from YEp51 plasmid, as aimilarly CPR (D33):pET15b expression vector was producedoning a 2.3 kb SalIBclI fragment containing CPR (D33)eased from CPR (D33):YEp51 (16), into the unique XhoIite of the E. coli expression vector, pET15b. All recombinantnipulations and bacterial transformations were carried outbed previously (17).

    pression and purification of native and soluble yeast CPRsli. Protein expression was carried out by following thee described previously (18). The expression vectors wereed into BL21DE3 (pLysS) strain. The transformed strains

    wn overnight at 37C and with shaking at 150 rpm in Luria) containing ampicillin (50 mg/ml) and chloramphenicol (34eterologous protein expression was induced with 0.5 mM

    l-b-D-thiogalactopyranoside (IPTG) for 20 h at 25C andking at 190 rpm after the cell density had reached to anensity of 0.50.7 at 660 nm. The cells were harvested at 4Cifugation at 5000g for 10 min. The cell pellet of 1 L culturespended in 40 ml of buffer A (50 mM KPi [pH 7.5] bufferg 0.5 M NaCl). Cells were lysed by freezethawing. The cell

    ere centrifuged at 4C for 10 min at 5000g to remove celld then at 100,000g at 4C for 60 min to separate membrane(pellet) from cytosolic fractions (supernatant). The mem-ctions were resuspended in buffer A. Protein concentrationrmined by the bicinchoninic acid method (BCA; Sigma)

    vine serum albumin standards.

    ation of native and soluble CPR. His6-tagged native andPR were purified by a single step using nickel-chelating

    chromatography. Membrane bound CPR was solubilized(w/v) sodium cholate as described previously (19). Both

    ed, native CPR and the cytosolic fraction containing solublere applied onto a 5 ml Ni21-NTA affinity chromatographyQiagen) previously equilibrated with 5 volumes of binding0 mM TrisHCl, pH 7.5, containing 500 mM NaCl and 50ine). The column was washed with 10 volumes of bindingd 10 volumes of wash buffer (binding buffer containing 20azole). Highly purified CPR protein was obtained when a

    adient of 5 to 100 mM imidazole (10 ml each of wash bufferion buffer) was used for elution. The fractions containingnative and soluble CPR (monitored by cytochrome c reduc-y) were pooled, dialyzed overnight against 10 mM potas-sphate buffer, pH 7.5, concentrated by ultrafiltration usingembrane (Whatman) to 30 mg/ml and stored at 280C. All

    re carried out at 4C and 1 ml/min flow rate. SDSPAGEormed as described by Laemmli (12). A set of SDSproteins (Sigma) were used and consisted of myosin (205 kDa),sidase (116 kDa), phosphorylase b (97 kDa), bovine serum(66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29e electrophoresed gel was stained with Coomassie Bluevisualize protein bands.

    (10).1U ofTo ththe rphospproprsuspepolyeaddedbatedimenNADmetaml (0hydrowater3 3GC/Mcolum(1 miInjectwas h

    DetCPR.whenwithtem csistedof gluml. Tthe vwhenbancetentsenzymstratechromtionsreducKinetthe MLineaconstperfo

    RES

    Exprin

    ThtheCPRthelocatWesyieldpresprotof EclearCPR

    49h reaction mixture contained 0.5 nmol purified CYP61 andrified native or soluble yeast CPR in a total volume of 50 ml.50 mg dilauroylphosphatidylcholine (DLPC) was added andtion volume adjusted to 950 ml with 100 mM potassiumte buffer, pH 7.2. Ergosta-5,7-dienol was added at the ap-

    concentration and the mixture sonicated until a whiteon had formed. Ergosta-5,7-dienol was purified from aresistant erg5 mutant of S. cerevisiae (20). NADPH wasthe mixture to start the reaction. All reactions were incu-37C for 20 min in a shaking water bath. In control exper-he involvement of CPR was examined by omission of P450,or CPR to the reconstituted system. Sterol substrate andte were extracted following the addition of 3 ml methanol, 2w/v) pyrogallol in methanol and 2ml 60% (w/v) potassium

    e (in water) and incubation at 90C for 2 h in a preheatedth. After cooling the saponified mixture was extracted withl hexane and dried under nitrogen. A Hewlett/Packardas used to confirm sterol identities. An Ultra 1 capillaryas used (10 m 3 0.2 mm) on a temperature program 50C

    ncreased by 40C/min to 290C with a run time of 17 min.port temperature was 280C (splitless) and the carrier gas

    um at 40 kPa.

    ination of the kinetic mechanism of native and soluble yeastinetics assays relied on the change in absorbance at 550 nm

    idized cytochrome c is converted into reduced cytochrome cextinction coefficient of 21.1 mM21. The kinetics assay sys-ained an enzymatic NADPH regeneration system and con-0.1 M TrisHCl, pH 7.8, 2 mM glucose-6-phosphate, 3 unitse-6-phosphate dehydrogenase (Sigma) in a total volume of 1cytochrome c concentration was 50 mM when NADPH wasble substrate and the NADPH concentration was 50 mMochrome c was the variable substrate. The change in absor-550 nm was monitored against time at 26C. Protein con-12 and 41 ng were used per assay for the native and soluble. To determine the enzyme mechanism of each CPR, sub-turation experiments were performed varying the cyto-concentration at three different fixed NADPH concentra-

    2.5, 5, and 50 mM. Velocities are expressed as picomolescytochrome c produced per minute per picomole of enzyme.arameters were determined by nonlinear regression usingaelisMenten equation to determine km and vmax values.

    egression was used to analyze the LineweaverBurk plotsted for the enzyme mechanism studies. The analyses wered using ProFit 5.01 (QuantumSoft, Zurich, Switzerland).

    TS

    sion of Native and Soluble Yeast CPR Proteinscoli

    yeast CPR contents were determined by usingochrome c reduction assay. As expected nativecalized in the membrane fraction of E. coli andino-terminal truncated and soluble CPR formin the cytosolic fraction (also confirmed by

    n blot, not shown). The specific contents andare shown in Table 1. Soluble CPR protein ex-

    at very high levels compared to the native CPR. No CPR was detected in the cytosolic fractionsoli expressing native CPR protein. The resultsindicated that the N-terminal truncated yeastcalized in the cytosol following expression in a

  • similarCPR fo

    PurificSolu

    SDS1) revenantlythe nat81 kDacatingobtainetive animal aNADPHincubaa decreance ofteristicoxidizetinguis23).

    Purifitheir aity of thwas 52with puorganisview, 6lated areduce

    SterolReco

    ErgoreconstCYP61uble Cdesatuenzymamin/nm20 mM

    ergosterol formed/minute/nmol CYP61 for sterol D22-desaturation driven by native CPR. No significant dif-

    cectiteasled

    tio

    ticlu

    thae

    me

    . 1miinimg

    TABLE 1

    The Specific Contents and Protein Yields of Native andSolublerichia c

    CPR

    NativeSoluble

    Vol. 286, No. 1, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSmanner to previous reports for mammalianllowing trypsin-cleavage experiments (21).

    ation and Characterization of Native andble Yeast CPR

    PAGE of purified native and soluble CPRs (Fig.aled the soluble enzyme consisted of predomi-(ca. 95%) one polypeptide of Mr 75 kDa and thative CPR contained one major polypeptide of Mr. Both purified CPRs were yellow in color, indi-the presence of flavin. Further confirmation wasd through UV/visible spectral analysis. The na-d soluble CPRs produced a spectrum with max-bsorption at 455 and 380 nm. Addition of

    to pure CPR followed by air reoxidation (byting at room temperature for 10 min) resulted inase in the absorption at 455 nm and the appear-a broad absorption band 550650 nm charac-of the air-stable semiquinone (not shown). The

    d and reduced spectra of both CPRs were indis-hable from those of other microsomal CPRs (22,

    ed native and soluble CPR were assayed bybility to reduce cytochrome c. The specific activ-e purified native CPR in reducing cytochrome cmmol/min/mg protein. This value is consistentblished values for CPRs purified from variousms, i.e., 5060 mmol/min/mg protein (for re-). The specific content of reductase was calcu-s 17 nmol/mg protein (by assuming 1 nmol CPRs 3 mmol of cytochrome c).

    D22-Desaturase (CYP61) Activity Followingnstitution with Native and Soluble CPR

    sta-5,7-dienol was aerobically metabolized by aituted monooxygenase system containingand either native and soluble yeast CPR. Sol-PR could drive CYP61 mediated sterol D22-ration giving a km of 25 mM and a maximaltic rate (vmax) of 3.1 nmol ergosterol formed/ol CYP61. These values compared with a km of

    and a maximal enzymatic rate (vmax) of 2.1 nmol

    ferenreduthe sby msteroshowreac

    KineSo

    BoMichchro

    FIGacrylaby staca. 5

    CPR Following Heterologous Expression in Esche-oli

    Amount (nmol)

    per mg protein

    per L cultureCytosol Membranes

    ND 0.02 6 0.005 7.5 6 2.10.35 6 0.06 ND 112 6 11.7

    50in km was observed coupled with only a slighton in CYP61 catalytic activity. The identity ofrol peaks in gas chromatography were confirmeds spectroscopy and showed conversion of the

    substrate into ergosterol. Control experimentsP450, CPR and NADPH dependency for the

    n.

    Mechanism Determination for Native andble Yeast CPR

    yeast native and soluble CPRs obeyedlisMenten kinetics with respect to both cyto-c and NADPH (Fig. 2). The kinetic parameters

    . SDSPAGE of native and soluble yeast CPRs. An 11%de resolvi...

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