Phanerochaete chrysosporium NADPH-cytochrome P450 reductase kinetic mechanism

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  • Phanerochaete chrysosporium NADPH-cytochrome P450reductase kinetic mechanism

    Andrew G.S. Warrilow, David C. Lamb, Diane E. Kelly, and Steven L. Kelly*

    Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, The University of Wales Aberystwyth, Aberystwyth SY23 3DA, UK

    Received 10 October 2002


    The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cyto-

    chromeP450 oxidoreductase (CPR;EC1.6.2.4) gene and>123 cytochromeP450 (CYP) genes.Howa singleCPRcandrivemanyCYPsis an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the

    cytochrome P450 catalytic system. Native CPR and a NH2-terminally truncated derivative lacking 23 amino acids have been over-

    expressed in Escherichia coli and puried to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity

    revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reecting dierences in

    CPRkineticmechanisms evenwithin a single kingdomof life. Removal of theN-terminal anchor ofP. chrysosporiumCPRdid not alter

    the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modication for structural studies.

    2002 Elsevier Science (USA). All rights reserved.

    Keywords: NADPH-cytochrome P450 oxidoreductase; Cytochrome P450; Phanerochaete chrysosporium; Purication; Kinetics; Reaction mechanism

    Eukaryotic cytochromes P450 (CYP) are membrane

    proteins usually located in the endoplasmic reticulum [1]

    and exhibit extraordinary diversity. Genome sequencing

    projects have shown approximately 57 CYPs in humans,

    90 in Drosophila melanogaster, 80 in Caenorhabditis

    elegans, and 276 in Arabidopsis thaliana, but only 3 in

    Saccharomyces cerevisiae []. The primary role of cytochromes

    P450 is the monooxygenation of diverse substrates of

    endogenous or exogenous origin [2]. NADPH-cyto-

    chrome P450 oxidoreductase (CPR; EC is a eu-

    karyotic membrane-bound avoprotein that is essential

    for the transfer of electrons during theCYP catalytic cycle

    in the endoplasmic reticulum. However, unlike CYP,

    CPR is encoded by a single gene with the exception of A.thaliana, where two are present [3]. Amino acid sequence

    homologies between CPRs from dierent organisms are

    higher in contrast to CYPs where only three residues are

    conserved. Hence, electron transfer from CPR to CYP is

    thought to reside in the overall electrostatic and hydro-

    phobic forces in the proteinprotein interaction [4]. The

    mechanism by which recognition and electron transfer

    proceed is an important area needing clarication and

    some biodiversity in this has already been observed [2,5].

    In the present study, we have probed the kinetic

    mechanism of CPR electron transfer in an organism with

    a high CYP complement (Phanerochaete chrysosporium)and considered our results with the CPR kinetic mecha-

    nism within a low CYP complement organism (S. cere-

    visiae). The recently completed genome sequence of the

    white rot fungusP. chrysosporium (as yet unpublished, see re-

    vealed the presence of one CPR gene, with a large pre-

    dicted molecular mass (>80 kDa) and >123 CYP genes.This is in contrast to the yeastS. cerevisiaewhere only oneCPR and three CYP genes were revealed. The present

    data indicate that P. chrysosporium CPR follows a dif-

    ferent kinetic mechanism from S. cerevisiae [6].

    Materials and methods

    Chemicals. All chemicals were obtained from Sigma Chemical

    Company (Poole, UK), unless otherwise stated.

    Biochemical and Biophysical Research Communications 299 (2002) 189195


    * Corresponding author. Fax: +44-1970-622350.

    E-mail address: (S.L. Kelly).

    0006-291X/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved.PII: S0006 -291X(02 )02600 -1

  • Cloning of native and soluble P. chrysosporium CPRs for expression

    in E. coli. A Lambda ZAP II cDNA library for P. chrysosporium

    ATCC24725 was constructed from total RNA extracted from 3-day-

    old cultures of P. chrysosporium ATCC24725 and reagents from

    Stratagene (LaJolla, California). PCR primers against the CPR gene

    sequence [7] incorporated a NdeI site in the forward primers and a 4-

    His tag and HindIII site in the reverse primer. Forward primers F1 (50-CAT CGC CAT ATG GCC GTA TCT TCG TCT TCG-30) and F2(50-CAT CGC CAT ATG CGC GAG CAA ATC TTC T-30) were usedto isolate CPR genes that were full-length (native) and D23 amino acidresidues, respectively. The reverse primer used, R1, was 50-ACT GCTAAG CTT CTA GTG ATG GTG ATG CGA CCA GAC ATC CAA

    CAA-30. PCR used an annealing temperature of 56 C for both CPRgenes. CPR products were rst cloned into pGEMT-Easy vector

    plasmid and sequenced. Authentic CPR genes were excised using di-

    gestion with NdeI/HindIII and then cloned into pET17b vector.

    Heterologous expression in E. coli, purication of native, and soluble

    P. chrysosporium CPRs. Expression was undertaken in E. coli

    BL21DE3 (pLysS). An overnight culture (5ml) was used to inoculate

    500ml of Terric-Broth (TB) containing ampicillin (100lg/ml).Following growth for 6 h at 37 C, 170 rpm, heterologous proteinexpression was induced with addition of 0.5mM isopropyl-b-DD-thiogalactopyranoside (IPTG) and incubated for 18 h, 120 rpm, at

    29 C. The cells were harvested by centrifugation at 5000g for 10min.All following procedures were carried out at 4 C. The cell pellet wasresuspended in 25ml of 100mM potassium phosphate, pH 7.5 buer.

    Cells were broken by passage through a C5 Emulsiex high pressure

    homogeniser operating at 170,000 kPa (Glen Creston, Stanmore,

    Middlesex). The cell lysates were centrifuged 10min at 5000g to re-

    move cell debris and then at 180,000g for 90min to separate membrane

    fractions (pellet) from cytosolic fractions (supernatant). The mem-

    brane fractions were suspended in 50mM potassium phosphate buer,

    pH 7.5, 20% glycerol. CPR activity was monitored by the reduction of

    cytochrome c. Background levels of reductase activity were determined

    using the cytosolic and membrane extracts from control cultures of

    E. coli that contained just the empty pET17b plasmid and these were

    then subtracted from the activities obtained with the CPR clones.

    Protein concentration was determined by the bicinchoninic acid

    method (BCA) using bovine serum albumin standards.

    Purication and characterisation of native and soluble CPR. Mem-

    brane bound full-length CPR was solubilised with 2% (w/v) sodium

    cholate as described previously [8] prior to purication. His4-tagged

    native and soluble D23 CPRs were puried by a single-step usingnickel-chelating anity chromatography as previously described [9].

    Fractions containing puried native and soluble CPRs (monitored by

    cytochrome c reduction assay) were pooled, dialysed overnight against

    20mM potassium phosphate buer, pH 7.5, 20% glycerol, and stored

    at )80 C until use. SDSPAGE was performed as essentially describedby Laemmli [10] using an 8% resolving gel. A set of SDSprotein

    standards (MW-SDS-200, Sigma) were used for calibration and Coo-

    massie blue R250 to visualise protein bands. The absolute absorption

    spectra were determined between 300 and 700 nm using native CPR

    (75lM) and D23 CPR (80 lM) in 1M TrisHCl, pH 8.1. NADPH-reduced absorption spectra of the two CPRs were determined in the

    presence of 0.2mM NADPH. These spectral determinations were

    made using a Hitachi U-3310 UV/Vis spectrophotometer (San Jose,


    Substrate saturation kinetics of native and soluble P. chrysospo-

    rium CPRs. Kinetics assays relied on the change in absorbance at

    550 nm at 25 C when oxidised cytochrome c is converted into re-duced cytochrome c with an extinction coecient of 21mM1 cm1

    [11] and contained an enzymatic NADPH regeneration system

    consisting of 0.2M TrisHCl, pH 7.8, 2mM glucose-6-phosphate,

    and 3U glucose-6-phosphate dehydrogenase in a total volume of

    1ml. Protein contents of 1.55 and 0.128lg were used per assay forthe native and soluble D23 enzyme, respectively. Substrate satura-tion experiments were performed varying the cytochrome c con-

    centration at ve dierent xed NADPH concentrations (1.5, 3, 6,

    12.5, and 50lM) and by varying the NADPH concentration atseven dierent xed cytochrome c concentrations (2.5, 5, 10, 20, 30,

    40, and 50 lM). Velocities are expressed as nmoles reduced cyto-chrome c produced per minute. Kinetic parameters were determined

    by non-linear regression using the MichaelisMenten equation to

    determine Km and Vmax values. Linear regression was used to analysethe EadieScatchard and LineweaverBurk plots constructed. A plot

    of (Km/Vmax) against the reciprocal of substrate concentration wasconstructed to determine Kd values for both NADPH and cyto-chrome c. Analyses were performed using ProFit 5.01 (Quantum-

    Soft, Zurich, Switzerland).

    Inhibition studies with NADP and reduced cytochrome c. Inhibitionassays were performed without the presence of the NADPH regener-

    ation system. Substrate saturation studies varying cytochrome c con-

    centration at a constant 50 lM NADPH were performed in thepresence and absence of 75 lM NADP using the two P. chrysospo-rium CPR enzymes. Substrate saturation studies varying NADPH

    concentration at a constant 50 lM oxidised cytochrome c were per-formed in the presence and absence of 45lM reduced cytochrome cand 75 lMNADP. Each kinetic determination was made in triplicateat a constant 25 C in 0.2M TrisHCl, pH 7.8. The relative reactivityof the two CPR enzymes towards NADH was determined by replacing

    the 50 lM NADPH in the non-regenerative assay system with 50lMNADH in the presence of 50lM oxidised cytochrome c.

    Immobilisation of the cytochrome coxD23 CPR complex and itsreduction by NADPH. Conjugation of oxidised cytochrome c to D23CPR was performed as by Nisimoto [12]. The cytochrome coxCPRconjugation protein was puried by using Sephadex G50SF and re-

    duced with 0.1mM NADPH in 0.1M TrisHCl, pH 8.1. Five micro-

    litres containing 1mM oxidised cytochrome c and 40lM D23 CPR wasapplied to PVDF membrane equilibrated in 0.1M TrisHCl, pH 8.1.

    On adsorption the membrane was washed with quenching buer

    (0.1M TrisHCl, pH 8.1, 0.2M NaCl, and 0.05% Tween 20) for 20min

    prior to blocking with 5% w/v dried skimmed milk powder in

    quenching buer for 30min. The membrane was washed again in

    quenching buer prior to equilibration in 0.1M TrisHCl, pH 8.1, for

    20min. Solid NADPH was added to a nal concentration of 2mM and

    the membrane was incubated at room temperature for a further 4 h.

    Control reactions in which NADP was used instead of NADPH andoxidised cytochrome c bound to the PVDF membrane instead of the

    cytochrome coxCPR mixture were also performed.

    Results and discussion

    Since there is only one CPR gene in eukaryotes with

    the exception of some plant species [13], CPR must be

    able to interact with and reduce the widely divergent

    cytochromes P450 that exist in each organism. Kinetic

    mechanisms and their diversity between and within or-

    ganisms is an important area of investigation, particu-larly where a huge number of CYPs are encountered in

    one organism as has been discovered in P. chrysospo-

    rium. There have been only limited previous investiga-

    tions of this type with rat liver CPR [14], housey CPR

    [15], and yeast CPR [6] and we have initiated this work

    as a beginning for biochemical studies on this P. chry-

    sosporium system. Here we describe the successful use of

    pET-based techniques for the expression of this type ofmembrane protein and the successful generation of full-

    length CPR and a soluble version that is currently the

    subject of crystallization trials.

    190 A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189195

  • Expression, purication, and characterisation of native

    and soluble P. chrysosporium CPRs

    Both CPRs were successfully expressed in E. coli

    without the need for modication of the N-terminus as

    is frequently undertaken for CYP expression. The NH2-

    terminal truncation site was chosen from a plot of polar

    free energies [16] of the amino acid residues (data notshown). The main N-terminal membrane anchor region

    appeared to be residues 224. Primers were designed so

    that full-length (native) and truncated cytosolic D23CPRs would be obtained and results conrmed the

    membrane anchor prediction with 96% of the overex-

    pressed D23 CPR being localised in the cytosolic frac-tion in contrast to the full-length CPR that was localised

    in the membrane. The D23 soluble CPR protein

    expressed at a higher level, of 4.7 lmol/L, compared tothe native CPR protein at just 0.84 lmol/L. Chroma-tography of the native P. chrysosporium and D23 trun-cated CPRs on Ni2-NTAAgarose resulted in 20-foldincreases in purity being obtained (Table 1) and were

    over 99% pure when analysed by SDSPAGE. The native

    enzyme and D23 CPRs had apparent Mr values of 88.2and 85.9 kDa, respectively, compared to the theoreticalMr values of 81.6 and 79.3 kDa. The puried native andD23 CPRs were both yellow in colour after elution fromthe Ni2-NTAAgarose column, indicating the presenceof avin. Further conrmation was obtained through

    UV/visible spectral analysis (Fig. 1). Both the native and

    D23 CPRs produced spectra that were typical of micro-somal CPRs [17,18] and were reduced in vitro by 0.2mM

    NADPH to form the air-stable neutral avin semi-qui-none, characterised by the broad absorbance peak at

    585 nm (Fig. 1). The specic activities of the puried

    native and D23 CPRs in reducing cytochrome c were3.3 and 14.8 lmol/min/mg protein, respectively.

    Kinetic mechanism determination for native and soluble

    P. chrysosporium CPRs

    Both native and D23 P. chrysosporium CPRs obeyedMichaelisMenten kinetics with respect to both cyto-

    chrome c and NADPH (Fig. 2) and gave similar kinetic

    parameters (Table 2). The Km values for cytochrome cwere 11 and 12 lM and the Km values for NADPH werenearly identical at 1.9 and 2.2 lM. The maximumturnover numbers were determined to be 19 and 42 for

    the native and D23 CPR enzymes, respectively. Thiscompares with Km values for cytochrome c of 1.6 lM foryeast CPR [6], 4.6 lM for housey CPR [15], and 3.4 lMfor rat CPR [14]. P. chrysosporium CPR has an 8-fold

    lower anity for cytochrome c than yeast CPR [6]. The

    Km values for NADPH were 12lM for yeast [6], rat[14], and housey [15] CPRs. EadieScatchard plots of

    the substrate saturation data (Fig. 2) gave a distinctive

    pattern of converging lines that met behind the S=v-axis. This result excludes the possibility that the CPRenzyme mechanism was bi bi ping pong, as such a

    mechanism would give a convergence of the lines at the

    S=v-axis at S 0. This kinetic pattern suggests a bi-reactant sequential mechanism in which both substrates

    must bind to the enzyme to form a ternary complex

    before the products can be formed and released. Such a

    Table 1

    The specic activties and yields of native and soluble CPR enzymes f...


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