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

Abstract

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 singleCPRcandrivemanyCYPs

is 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 purified 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 reflecting differences 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 modification for structural studies.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: NADPH-cytochrome P450 oxidoreductase; Cytochrome P450; Phanerochaete chrysosporium; Purification; 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 [http://drnelson.utmem.edu/CytochromeP450.html]. 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 1.6.2.4) is a eu-

karyotic membrane-bound flavoprotein 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 different 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 protein–protein interaction [4]. The

mechanism by which recognition and electron transfer

proceed is an important area needing clarification 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

http://drnelson.utmem.edu/CytochromeP450.html) 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) 189–195

www.academicpress.com

BBRC

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

E-mail address: steven.kelly@aber.ac.uk (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 used

to isolate CPR genes that were full-length (native) and D23 amino acid

residues, respectively. The reverse primer used, R1, was 50-ACT GCT

AAG CTT CTA GTG ATG GTG ATG CGA CCA GAC ATC CAA

CAA-30. PCR used an annealing temperature of 56 �C for both CPR

genes. CPR products were first 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, purification 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 Terrific-Broth (TB) containing ampicillin (100lg/ml).

Following growth for 6 h at 37 �C, 170 rpm, heterologous protein

expression 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 was

resuspended in 25ml of 100mM potassium phosphate, pH 7.5 buffer.

Cells were broken by passage through a C5 Emulsiflex 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 buffer,

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.

Purification 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 purification. His4-tagged

native and soluble D23 CPRs were purified by a single-step using

nickel-chelating affinity chromatography as previously described [9].

Fractions containing purified native and soluble CPRs (monitored by

cytochrome c reduction assay) were pooled, dialysed overnight against

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

at )80 �C until use. SDS–PAGE was performed as essentially described

by Laemmli [10] using an 8% resolving gel. A set of SDS–protein

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 Tris–HCl, 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,

California).

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 coefficient of 21mM�1 cm�1

[11] and contained an enzymatic NADPH regeneration system

consisting of 0.2M Tris–HCl, 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 for

the native and soluble D23 enzyme, respectively. Substrate satura-

tion experiments were performed varying the cytochrome c con-

centration at five different fixed NADPH concentrations (1.5, 3, 6,

12.5, and 50lM) and by varying the NADPH concentration at

seven different fixed 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 Michaelis–Menten equation to

determine Km and Vmax values. Linear regression was used to analyse

the Eadie–Scatchard and Lineweaver–Burk plots constructed. A plot

of (Km/Vmax) against the reciprocal of substrate concentration was

constructed 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 NADPþ and reduced cytochrome c. Inhibition

assays 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 the

presence and absence of 75 lM NADPþ 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 c

and 75 lM NADPþ. Each kinetic determination was made in triplicate

at a constant 25 �C in 0.2M Tris–HCl, pH 7.8. The relative reactivity

of 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 cox–D23 CPR complex and its

reduction by NADPH. Conjugation of oxidised cytochrome c to D23CPR was performed as by Nisimoto [12]. The cytochrome cox–CPRconjugation protein was purified by using Sephadex G50SF and re-

duced with 0.1mM NADPH in 0.1M Tris–HCl, pH 8.1. Five micro-

litres containing 1mM oxidised cytochrome c and 40lM D23 CPR was

applied to PVDF membrane equilibrated in 0.1M Tris–HCl, pH 8.1.

On adsorption the membrane was washed with quenching buffer

(0.1M Tris–HCl, 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 buffer for 30min. The membrane was washed again in

quenching buffer prior to equilibration in 0.1M Tris–HCl, pH 8.1, for

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

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

Control reactions in which NADPþ was used instead of NADPH and

oxidised cytochrome c bound to the PVDF membrane instead of the

cytochrome cox–CPR 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], housefly 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) 189–195

Expression, purification, and characterisation of native

and soluble P. chrysosporium CPRs

Both CPRs were successfully expressed in E. coli

without the need for modification 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 2–24. Primers were designed so

that full-length (native) and truncated cytosolic D23CPRs would be obtained and results confirmed 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 to

the native CPR protein at just 0.84 lmol/L. Chroma-

tography of the native P. chrysosporium and D23 trun-

cated CPRs on Ni2þ-NTA–Agarose resulted in 20-fold

increases in purity being obtained (Table 1) and were

over 99% pure when analysed by SDS–PAGE. The native

enzyme and D23 CPRs had apparent Mr values of 88.2

and 85.9 kDa, respectively, compared to the theoreticalMr values of 81.6 and 79.3 kDa. The purified native and

D23 CPRs were both yellow in colour after elution from

the Ni2þ-NTA–Agarose column, indicating the presence

of flavin. Further confirmation 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 flavin semi-qui-none, characterised by the broad absorbance peak at

585 nm (Fig. 1). The specific activities of the purified

native and D23 CPRs in reducing cytochrome c were

3.3 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 obeyedMichaelis–Menten kinetics with respect to both cyto-

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

parameters (Table 2). The Km values for cytochrome c

were 11 and 12 lM and the Km values for NADPH were

nearly identical at 1.9 and 2.2 lM. The maximum

turnover numbers were determined to be 19 and 42 for

the native and D23 CPR enzymes, respectively. This

compares with Km values for cytochrome c of 1.6 lM foryeast CPR [6], 4.6 lM for housefly CPR [15], and 3.4 lMfor rat CPR [14]. P. chrysosporium CPR has an 8-fold

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

Km values for NADPH were 1–2lM for yeast [6], rat

[14], and housefly [15] CPRs. Eadie–Scatchard plots of

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

pattern of converging lines that met behind the ½S�=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

½S�=v-axis at ½S� ¼ 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 specific activties and yields of native and soluble CPR enzymes following heterologous expression in Escherichia coli

CPR enzyme Specific activity (lmol/min/mg protein) Increase in purity

(Fold)

Amount

(lmol/L)aCytosol Membranes Nickel column

Native 0.034 0.162 3.265 20.2 0.84

D23 0.746 0.169 14.76 19.8 4.67

a Litre of culture.

Fig. 1. The absorption spectra of purified P. chrysosporium native CPR

(A) and cytosolic D23 (B) CPR. Absorption spectra of the native CPR

((A) 75 lM) and the cytosolic D23 CPR ((B) 80 lM) were determined

both in the absence (solid line) and presence of 0.2mM NADPH

(dashed line).

A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189–195 191

pattern of intersecting lines behind the ½S�=v-axis indi-

cates that the Km is greater than the Kd and has been

characterised for mechanisms that involve �sticky�susbstrates [19]. The Kd values were 1.5- to 2-fold lower

than the Km values (Table 2). Inhibition studies using45 lM reduced cytochrome c proved unsuccessful in

significantly inhibiting the CPR reaction when NADPH

was the variable substrate. The presence of 45 lM of

reduced cytochrome c in an assay containing 37.5 lMNADPH and 50 lM oxidised cytochrome c caused ob-

served inhibitions of 8% and 11% for the native and D23CPRs, respectively. Substrate saturation experiments

varying oxidised cytochrome c and NADPH concen-trations individually in the presence or absence of 45 lMreduced cytochrome c gave no discernable inhibition

(data not shown). The lack of inhibition of CPR by

Fig. 2. Initial velocity patterns obtained from enzyme mechanism substrate saturation experiments. Substrate saturation experiments using

P. chrysosporium native and soluble D23 CPRs with increasing cytochrome c concentrations were performed at five fixed NADPH concentrations of

50lM (d), 12.5lM (j), 6lM (N), 3lM (�), and 1.5 lM (s). Eadie–Scatchard plots of these data were constructed for native (A) and soluble D23(B) CPRs. Substrate saturation experiments with increasing NADPH concentrations were performed at seven fixed cytochrome c concentrations of

50lM (d), 40lM (j), 30 lM (N), 20lM (s), 10 lM (�), 5lM (M), and 2.5 lM ( ). Eadie–Scatchard plots of these data were constructed for

native (C) and soluble D23 (D) CPRs. The data points at 50mM have been omitted to aid clarity in interpreting the kinetic pattern but these data

points were used in the kinetic fitting process. Velocities (v) are expressed as nmoles reduced cytochrome c produced per minute. All data points are

means of three replicates.

192 A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189–195

reduced cytochrome c prevents the identification of theorder of substrate binding by traditional kinetic meth-

ods. Therefore both ordered and random sequential

kinetic mechanisms can be considered for P. chrysos-

porium CPR.

If substrate binding to CPR is random, oxidised cy-

tochrome c should be able to bind to both oxidised and

reduced forms of CPR and NADPH should be able to

bind to the free CPR enzyme and to the cytochrome cox–CPR complex. To establish the absolute binding order

of substrates to CPR a conjugated cytochrome cox–CPRprotein was generated. The isolated conjugated protein

was reduced by 0.1mM NADPH, giving an absorbance

Table 2

Kinetic constants derived for the P. chrysosporium CPR enzymes

Kinetic constant Native D23

Vmax (nmol/min) 4:08� 0:12 4:01� 0:16

Km for NADPH (lM) 1:92� 0:03 2:20� 0:13

Km for cytochrome c (lM) 11:31� 0:34 11:54� 0:29

Kd for NADPH (lM) 1.5 1.2

Kd for cytochrome c (lM) 8.3 5.8

Ki for NADPþ (lM) 22:2� 0:9 20:6� 1:2

Turnover number (s�1)

(NADPH)

18.6 41.5

Turnover number (s�1)

(NADH)

0.12 0.89

Fig. 3. Initial velocity patterns obtained by inhibition of P. chrysosporium CPR enzymes with NADPþ. Substrate saturation experiments were

performed using native (A) and soluble D23 (B) CPR enzymes with increasing cytochrome c concentrations in the absence () and the presence (s)

of 75lM NADPþ. Substrate saturation experiments were also performed using native (C) and soluble D23 (D) CPR enzymes with increasing

NADPH concentrations in the absence (d) and the presence (s) of 75 lM NADPþ.

A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189–195 193

peak at 550m. The cytochrome cox–D23 CPR compleximmobilised on PVDF membrane was also reduced by

NADPH, resulting in a colour change from red to pink

after 2 h and eventually to a pinkish yellow after 4 h. The

two controls remained in dark red colour after 4 h of

incubation. Therefore, NADPH can bind to and reduce

the cytochrome cox–D23 CPR complex as well as binding

to the free D23 CPR enzyme (Fig. 1). The D23 CPR

enzyme behaved in an identical fashion to the full-lengthCPR. The rapid reduction of free CPR with NADPH

(Fig. 1) indicates that NADPH can bind to the free CPR

enzyme. Preincubation of CPR with NADPH prior to

the addition of oxidised cytochrome c resulted in no

reduction in the observed CPR activity. This indicated

that oxidised cytochrome c can bind to the reduced CPR

enzyme (CPR–NADPH complex). A similar preincu-

bation of the CPR enzyme with oxidised cytochrome cprior to the addition of NADPH also resulted in no

reduction of the observed CPR activity. Therefore the

binding of both substrates of P. chrysosporium CPR is

random. There was no difference in the kinetic mecha-

nism between the native and D23 soluble CPRs, indi-

cating that the N-terminal membrane anchor did not

modulate the kinetic activity of P. chrysosporium CPR

towards cytochrome c or NADPH in vitro. ThereforeD23 CPR can be used as a valid model for future

structural studies of P. chrysosporium CPR.

If such a random bireactant sequential mechanism

involved no dead-end complexes then each product

would behave as a competitive inhibitor of each sub-

strate [19]. This was not the case with P. chrysosporium

CPR as the product NADPþ was a competitive in-

hibitor of NADPH, but was a mixed-type inhibitor ofoxidised cytochrome c (Fig. 3). Such an inhibition

pattern can be obtained if the CPR enzyme can form

dead-end complexes with one or both of its products.

Such dead-end complexes would be (CPR–Cyt.cox–NADPþ) and (CPR–NADPH–Cyt.cred) [19]. This in-

hibition pattern is not encountered with bireactant ping

pong mechanisms. Native and D23 CPR enzymes had

similar affinities for NADPþ with KiNADP values of 22and 21 lM and had a 5-fold lower affinity for NADPþ

than for the substrate NADPH. Both CPR enzymes

were insensitive to inhibition by reduced cytochrome c,

suggesting an extremely low affinity (high Km values)

for the reduced form. Therefore the rate of formation

of the (CPR–NADPH–Cyt.cred) dead-end complex

would be significantly lower than the rate of formation

of (CPR–Cyt.cox–NADPþ).Both P. chrysosporium CPR enzymes could utilise

NADH to reduce cytochrome c, however, at much re-

duced rates of 0.6% and 2.1% of those observed when

NADPH was the substrate for the native and D23 CPRs,

respectively. The observed rates with NADH were too

low to perform the same kinetic experiments that had

been previously performed with NADPH.

Rat liver CPR [14] was found to obey a two-site ping-pong kinetic mechanism, with site one responsible for

the NADPH/NADPþ half-cycle and site 2 responsible

for the Cyt.cox/Cyt.cred half-cycle. At high ionic strength

(0.85M) the cytochrome c half-cycle is a tetra uni ping-

pong mechanism and at lower ionic strength (0.20–

0.75M) it becomes a random sequential mechanism in

which NADPþ release occurs before the binding of cy-

tochrome c. Therefore rat liver CPR appears to be ahybrid between a traditional ping-pong mechanism and

a random sequential mechanism. Yeast CPR [6] was

found to display the kinetic characteristics of a standard

random bi bi ping-pong mechanism with a Lineweaver–

Burk plot of the substrate saturation experiments at

different fixed concentrations of the second substrate

resulting in a series of parallel lines. Housefly CPR [15]

was found to obey a bireactant random sequential ki-netic mechanism with both substrates having to bind to

the CPR enzyme to form a ternary complex prior to

product formation and release. Therefore the P. chry-

sosporium CPR kinetic mechanism is closer to that de-

scribed for housefly [15], and unlike yeast [6] or rat CPR

[14]. As the CPR kinetic mechanism varies between

different species this suggests that divergent evolution

has taken place, even within the fungi. The present workon the purification and characterisation of P. chrysos-

porium CPR has paved the way for the investigation of

activity of the remarkable CYP complement (CYPome)

of this organism.

Acknowledgment

We are grateful to the Biotechnology and Biological Science Re-

search Council of the United Kingdom for support.

References

[1] T.D. Porter, M.J. Coon, Cytochrome P450—multiplicity of

isoforms, substrates and catalytic and regulatory mechanisms,

J. Biol. Chem. 266 (1991) 13469–13472.

[2] D.C. Lamb, D.E. Kelly, N.J. Manning, M.A. Kaderbhai, S.L.

Kelly, Biodiversity of the P450 catalytic cycle: yeast cytochrome

b5/NADH cytochrome b5 reductase complex drives the entire

sterol 14-demethylation (CYP51) reaction, FEBS Lett. 462 (1999)

283–288.

[3] M. Mizutani, D. Ohta, Two isoforms of NADPH: cytochrome

P450 reductase in Arabidopsis thaliana—gene structure, heterolo-

gous expression in insect cells, and differential regulation, Plant

Physiol. 116 (1998) 357–367.

[4] I.F. Sevrioukova, H.Y. Li, H. Zhang, J.A. Peterson, T.L. Poulos,

Structure of a cytochrome P450–redox partner electron-transfer

complex, Proc. Natl. Acad. Sci. USA 96 (1999) 1863–1868.

[5] P.A. Williams, J. Cosme, V. Sridhar, E.F. Johnson, D.E. McRee,

Mammalian microsomal cytochrome P450 monooxygenase: struc-

tural adaptations for membrane binding and functional diversity,

Mol. Cell 5 (2000) 121–131.

[6] D.C. Lamb, A.J.S. Warrilow, K. Venkateswarlu, D.E. Kelly, S.L.

Kelly, Activities and kinetic mechanisms of native and soluble

194 A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189–195

NADPH-cytochrome P450 reductase, Biochem. Biophys. Res.

Commun. 286 (2001) 48–54.

[7] J.S. Yadav, J.C. Loper, Cytochrome P450 oxidoreductase gene

and its differentially terminated cDNAs from the white rot fungus

Phanerochaete chrysosporium, Curr. Genet. 37 (2000) 65–73.

[8] K. Venkateswarlu, D.C. Lamb, D.E. Kelly, N.J. Manning, S.L.

Kelly, The N-terminal domain of yeast NADPH-cytochrome

P450 reductase is not required in sterol biosynthesis, J. Biol.

Chem. 273 (1998) 4492–4496.

[9] A. Bellamine, A.T. Mangla, W.D. Nes, M.R. Waterman, Char-

acterization and catalytic properties of the sterol 14a-demethylase

from Mycobacterium tuberculosis, Proc. Natl. Acad. Sci. USA 96

(1999) 8937–8942.

[10] U.K. Laemmli, Cleavage of structural proteins during the

assembly of the head of bacteriophage T4, Nature 227 (1970)

680–685.

[11] S.D. Black, M.J. Coon, Structural features of liver microsomal

NADPH cytochrome P450 reductase: hydrophobic domain,

hydrophilic domain and connecting region, J. Biol. Chem. 257

(1982) 5929–5938.

[12] Y. Nisimoto, Localization of cytochrome c-binding domain on

NADPH-cytochrome P450 reductase, J. Biol. Chem. 261 (1986)

14232–14239.

[13] I. Benveniste, A. Lesto, M.P. Hasenfratz, G. Kochs, F. Durst,

Multiple forms of NADPH-cytochrome P-450 reductase in higher

plants, Biochem. Biophys. Res. Commun. 177 (1991) 105–112.

[14] D.S. Sem, C.B. Kasper, Effect of ionic strength on the kinetic

mechanism and relative rate limitation of steps in the model

NADPH cytcohrome P450 oxidoreductase reaction with cyto-

chrome c, Biochemistry 34 (1995) 12768–12774.

[15] M.B. Murataliev, A. Arino, V.M. Guzov, R. Feyereisen, Kinetic

mechanism of cytochrome P450 reductase from the housefly

(Musca domestica), Insect Biochem. Mol. Biol. 29 (1999) 233–242.

[16] D.M. Engelman, T.A. Steitz, A. Goldman, Identifying non-polar

transbilayer helices in amino acid sequences of membrane

proteins, Ann. Rev. Biophys. Biol. 15 (1986) 321–353.

[17] G.O. Kurzban, J. Howarth, G. Palmer, H.W. Strobel, NADPH-

cytochrome P-450 reductase: physical properties and redox

behavior in the absence of the FAD moiety, J. Biol. Chem. 265

(1990) 12272–12279.

[18] D.D. Oprian, M.J. Coon, Oxidation–reduction states of FMN

and FAD in NADPH-cytochrome P-450 reductase during reduc-

tion by NADPH, J. Biol. Chem. 257 (1982) 8935–8944.

[19] I.H. Segel, in: Enzyme Kinetics—Behavior and Analysis of Rapid

Equilibrium and Steady-State Enzyme Systems, Wiley, New York,

1993, pp. 274–309.

A.G.S. Warrilow et al. / Biochemical and Biophysical Research Communications 299 (2002) 189–195 195