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A new cytochrome P450 belonging to the 107L subfamily is responsible for theefficient hydroxylation of the drug terfenadine by Streptomyces platensis

Murielle Lombard a,⇑, Isabelle Salard b, Marie-Agnès Sari a, Daniel Mansuy a, Didier Buisson c

a Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR 8601, Université Paris Descartes, 45 rue des Saints-Pères, 75 270 Paris Cedex 06, Franceb Laboratoire Analyse et Modélisation pour la Biologie et l’Environnement, LAMBE, CNRS UMR 8587, Université Evry, boulevard François Mitterand, 91025 Evry Cedex, Francec Unité Molécules de Communication et Adaptation des Microorganismes, CNRS/MNHN FRE 3206, Muséum National d’Histoire Naturelle, 57 rue Cuvier, 75 005 Paris Cedex 05, France

a r t i c l e i n f o

Article history:

Received 4 November 2010

and in revised form 10 January 2011

Available online xxxx

Keyword:

Xenobiotics

Oxidation

Antihistamine

Actinomycete

Biotransformation

Ferredoxin reductase

CYP107L

P450terf

CYP2J2

PikC

Macrolides

a b s t r a c t

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative bio-

transformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reac-

tions. We report here the purification and identification of the enzyme responsible for the first step, a

cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450 terf , was

found to catalyze the hydroxylation of the t -butyl group of terfenadine and exhibited UV–Vis character-

istics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as

expected for the formation of a P450–substrate complex. In combination with spinach ferre-

doxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxyl-

ation of terfenadine and some of its analogues, such as terfenadone and ebastine, with km values at the

lM level, and kcat values around 30 minÀ1. Sequencing of the p450terf gene led to a 1206 bp sequence,

encoding for a 402 aminoacid polypeptide exhibiting 56–65% identity with the P450s from the 107L fam-

ily. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechno-

logical production of secondary metabolites, such as antibiotics or antitumor compounds, and in the

oxidative biotransformation of xenobiotics, such as drugs.

Ó2011 Elsevier Inc. All rights reserved.

Introduction

The availability of drug metabolites is crucial for drug develop-

ment and the current methods for producing these metabolites are

often slow and expensive. They rely upon the use of liver micro-

somes or recombinant enzymes, in particular cytochromes P4501

(P450s), that are half-life limited and costly, or upon chemical syn-

thesis that may also be expensive. Many efforts have been made

recently in the field of microbial transformations that produce

metabolites of xenobiotics [1].

Fexofenadine, the pharmacologically active metabolite of ter-

fenadine, is a H1 receptor antagonist and a second-generation anti-

histamine drug prescribed in allergic inflammations [2]. In man,

terfenadine undergoes extensive first-pass metabolism due to

cytochrome P450-dependent enzymatic activities (Fig. 1). Two oxi-

dation reactions are involved, i.e., an oxidative N -dealkylation lead-

ing to azacyclonol, that is mainly catalyzed by CYP3A4, and a

hydroxylation of the t -butyl group leading to hydroxyterfenadine,

which is mainly catalyzed by CYP2J2 but also by CYP4F12, CYP3A4

and CYP2D6 [3–8]. Hydroxyterfenadine undergoes subsequent

CYP2J2-dependent oxidation into the corresponding carboxylic

acid, fexofenadine, the active metabolite. The prodrug terfenadine

was superseded by fexofenadine several years ago, because of the

cardiotoxicity of terfenadine at high doses [9]. However, despite

structural similarities of these two compounds, the synthetic route

used to prepare terfenadine was found to be poorly efficient for

fexofenadine synthesis and gave very low yields (<10%) [10–12].

Moreover, oxidation of terfenadine by chemical methods mainly

led to N -oxidation-derived products, such as azacyclonol, a mole-

cule formerly used as an ataractive drug. Therefore, an efficient

method of direct transformation of terfenadine into fexofenadine

would be of particular interest for pharmaceutical industry.

We have previously demonstrated that Streptomyces platensis

NRRL 2364 efficiently biotransforms terfenadine into fexofenadine

[13,14]. This bioconversion is the result of three consecutive

oxidation reactions: (i) a hydroxylation of a methyl group from the

t -butyl moiety of terfenadine to give the primary alcohol, hydroxy-

terfenadine, (ii) an oxidation of the alcohol function into the corre-

sponding aldehyde, and (iii) an oxidation of the aldehyde to the

corresponding carboxylic acid, fexofenadine.

0003-9861/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.abb.2011.01.008

⇑ Corresponding author.

E-mail address: [email protected](M. Lombard).1 Abbreviations used: CYP or P450, cytochrome P450; Fd, ferredoxin; FdR, ferre-

doxin:NADP(+) oxidoreductase; HPLC-ESI-MS, high pressure liquid chromatography

electrospray ionization mass spectrometry; UV–Vis, UV–Visible spectroscopy; PVDF,

polyvinylidene difluoride; PMF, peptide mass fingerprint.

Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx

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Please cite this article in press as: M. Lombard et al., Arch. Biochem. Biophys. (2011), doi: 10.1016/j.abb.2011.01.008

http://dx.doi.org/10.1016/j.abb.2011.01.008

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In vivo terfenadine bioconversion studies with S. platensis whole

cells have shown that: (i) the three successive oxidation reactions

required molecular oxygen O2, (ii) terfenadine and hydroxyterfena-

dine biotransformation under 18O2-enriched atmosphere led to18O-labelled fexofenadine, (iii)additionof usual P450inhibitors, such

as clotrimazole and fluconazole, inhibited terfenadine oxidation, and

(iv) addition of soybean peptones enhanced fexofenadine formation

[15,16]. In thatregard,it is noteworthythatgenistein, an isoflavonoid

component of soybean flours, was previously shown to induce the

expression of a cytochrome P450, CYP105D1, also named P450soy, in

Streptomyces griseus [17,18]. The above results strongly suggested

that the oxidation of terfenadine into fexofenadine should involve

one or several P450-dependent monooxygenase(s).

To date, about 600 CYP genes from some 80 Streptomyces fila-

mentous bacteria have been reported in all searchable databases[19]. Some of the corresponding P450s are involved in the biosyn-

thesis of secondary metabolites, such as antibiotics, by the bacte-

ria; however, for most of them, no enzymatic activity has been

reported so far. Moreover, very few data are presently available

on the ability of those P450s to act as biocatalysts for the oxidation

of xenobiotics such as drugs, e.g., the oxidation of 7-ethoxycouma-

rin, precocene II, benzo[a]pyrene and warfarin by CYP105D1 from

S. griseus [18,20].

In an effort to find P450s from filamentous bacteria that would

be new biotechnological tools for the oxidative bioconversion of

xenobiotics, it was interesting to characterize the enzyme respon-

sible for the efficient and regioselective oxidation of terfenadine by

S. platensis. This article reports the isolation, purification and char-

acterization of the P450, called P450terf , that is responsible for thisreaction. Determination of its amino acid sequence showed that it

belongs to the 107L subfamily.

Materials and methods

Biochemicals and chemicals

Yeast extract, malt extract, glucose and agar were purchased

from Difco (Detroit, Mich., USA). Soybean peptone was purchased

from Organotechnie (La Courneuve, France). NADPH, cytochrome

c , glucose 6-phosphate dehydrogenase, leupeptin, chicken egg

lysozyme, terfenadine, spinach ferredoxin:NADP+ oxidoreductase

and spinach ferredoxin were purchased from Sigma–Aldrich (St

Quentin Fallavier, France). Deoxyribonuclease I (DNase I), aprotininand pepstatin were purchased from Euromedex (Souffelweyers-

heim, France). Ebastine was provided by Pharmapharm (Paris,

France). Terfenadone was synthesized as previously described

[21]. Kod DNA polymerase from Thermococcus kodakaraensis, and

detergent-based Bug Buster were from Novagen (Merck Chemicals

Ltd., Nottingham, UK).

Bacterial strain and growth conditions

Stock cultures were maintained on 2% malt extract agar and

stored at 4 °C. S. platensis cells (20 L) were aerobically grown at

30 °C, in YM (yeast extract 4 g/L, malt extract 10 g/L) or YMS med-

ium (Yeast extract 4 g/L, Malt extract 10 g/L, Soybean peptone 5 g/

L) in the presence of glucose (16 g/L), in a 25-L incubator (BiostatÒ

C, B. Braun Biotech International, Melsungen, Germany), with vig-

orous stirring (600 rpm). After 48 h culture, cells were harvested

and collected by continuous centrifugation (7000 rpm, 4 °C) and

stored at À80 °C.

Cytosolic and membrane extracts preparation

Cells (400 g) were washed in 200 mL cold 50 mM Tris–HCl buf-

fer, pH 7.6, containing 1 mM EDTA and 10% glycerol (TEG buffer),

and then resuspended in 3% of the original culture volume in the

same buffer containing chicken egg lysozyme (4 g) and DNase I

(1 mg). After 2 h incubation at 30 °C, cells were disrupted by addi-

tion of detergent-based Bug Buster reagent (0.5Â), in the presence

of protease inhibitors (0.5 mg leupeptin, 1 mg aprotinin, and 7 mg

pepstatin). All the following steps were performed at 4°C. Cellswere sonicated (10 Â 10 s, amplitude 40%, Vibracell 75115, Fisher

Bioblock Scientific, Illkirch, France) and centrifuged at 6000 g for

30 min to remove unbroken cells and debris. The cloudy superna-

tant, which contained both cytosolic and membrane fractions, was

then fractionated by centrifugation at 100,000 g for 1 h (Beckman

Ti50.2 rotor, Beckman ultracentrifuge). The supernatant containing

soluble proteins was collected and used for further purification.

The pellet containing membrane proteins was resuspended in

TEG buffer. Protein concentration of both cytosolic and membrane

fractions was estimated using the Bradford method [22].

Cytochrome P450 purification

Fractional precipitation of the proteins with ammonium sulfatewas performed at 40% and 80% (w/v). The 80% ammonium sulfate

terfenadine

azacyclonol

+

hydroxyterfenadine

fexofenadine(antihistamine drug)

O

H OH

(or CYP2J2, 2D6, 4F12, 3A4)

Streptomyces platensis

Streptomyces platensis

(or CYP2J2)

CYP3A4

NOH

OH

NHOH

NOH

OHOH

NOH

OH

O

OH

Fig. 1. Oxidation of terfenadine to fexofenadine by Streptomyces platensis and metabolism of terfenadine by human liver P450s.

2 M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx

Please cite this article in press as: M. Lombard et al., Arch. Biochem. Biophys. (2011), doi:10.1016/j.abb.2011.01.008





pellet was resuspended in TEG buffer and loaded onto a gel filtra-

tion column (HiPrep26/60Sephacryl S-200 HR, 320 mL, GE Health-

care, Amersham, Saclay, France) under the control of a Biologic

(Bio-Rad, Marnes-la-Coquette, France) device equipped with a

258 nm detector. Elution was done at 1 mL/min. Active fractions

were pooled and loaded onto an anion exchange column (UnoQ,

6 mL, Bio-Rad) in TEG buffer. A 20 mL linear gradient from 0 to

0.5 M NaCl, 1 mL/min, was applied. P450terf was eluted at250 mM NaCl. Active fractions were collected, concentrated with

Amicon YM30 centricon (Millipore, Guyancourt, France), equili-

brated with TEG buffer containing 30% ammonium sulphate and

loaded onto a hydrophobic column (Phenyl Sepharose CL4-B,

4 mL, GE Healthcare). An ammonium sulfate decreasing gradient

from 30% to 0% was applied. P450terf was eluted after the gradient

reached 0% ammonium sulfate. Active fractions were collected,

concentrated and loaded onto a gel filtration column (Superdex

75, HiLoad 16/60, 120 mL, GE Healthcare). P450terf active fractions

were collected, concentrated and analyzed by SDS PAGE and

UV–Vis spectroscopy.

Protein analysis

Protein concentration was determined according to the Brad-

ford method [22]. SDS–PAGE was performed by the protocol of Lae-

mmli using 12% polyacrylamide gels (Bio-Rad). UV–Vis spectra

were recorded with an Uvikon 930 spectrophotometer (Kontron

Instruments), in cells of 1 cm path length, at 25 °C. N-Terminal

amino acid sequencing was performed on purified P450terf by Euro-

gentec (Angers, France). The N-terminal sequence was determined

using an Applied Biosystem protein sequencer. Peptide mass fin-

gerprint using nano HPLC–ESI-MS/MS was performed on purified

P450terf by Proteome Factory (Proteome Factory AG, Berlin, Ger-

many). MS spectra were recorded with Esquire 3000, according

to the manufacturer’s instrument settings. The protein was identi-

fied using MS/MS ion search of Mascot search engine (Matrix Sci-

ence, London, England, http://www.matrixscience.com).

Cytochrome P450 activity assays

Terfenadone hydroxylation activity

Activity assays along the protein purification procedure were

performed by measuring hydroxylation of terfenadone (Fig. 2), a

terfenadine derivative whose hydroxylation was much more easily

followed than that of terfenadine itself, by HPLC coupled to UV

spectroscopy because of its much stronger absorbance at 254 nm

due to its conjugated Ar–C@O moiety. Reactions were carried out

at 28 °C in 0.1 M potassium phosphate buffer, pH 7.4, containing

1 mM NADPH, 1 lM spinach ferredoxin NADP+ oxidoreductase,

4 lM spinach ferredoxin, 100lM terfenadone and 0.1–200lg pro-

tein fractions. The reaction mixtures (0.2 mL) were incubated for

30 min, and reactions were stopped by addition of one volume of

methanol. After centrifugation, the samples were analyzed on a

Gilson HPLC apparatus (Villiers-le-Bel, France) equipped with a re-

verse-phase column Extend C18 (Agilent, 5 lm, 250Â 4.6, A.I.T.,

Interchim, Montluçon, France) and a Shimadzu-SDP A detector(k = 254 nm). HPLC–ESI-MS/MS experiments were done with a Sur-

veyor-LCQ Advantage mass spectrometer, with electrospray ionisa-

tion (ESI) negative mode, using a 4 kV capillary tube voltage and an

inlet temperature of 275 °C. HPLC experiments used a linear gradi-

ent of 10 mM ammonium acetate in water–acetonitrile (7:3–1:9)

at a flow of 0.25 ml/min and UV detection at 230 nm and 254 nm.

Enzymatic kinetic parameters

To determine the K m and kcat values of the P450terf catalyzed oxi-

dations, the reaction mixtures, in 0.2 mL 0.1 M phosphate buffer,

pH 7.4, contained P450terf (5 nM), NADPH (1 mM), spinach ferre-

doxin NADP+ oxidoreductase (1lM), spinach ferredoxin (2 lM),

varying amounts of terfenadone or its analogues (0.2–250 lM)

and 2% DMSO for substrate solubilization. After incubation for

5 minat 28 °C, the reaction was stopped by the addition of one vol-

ume of methanol, and analyzed by HPLC using the above described

procedure. It is noteworthy that the hydroxylation of terfenadone

and analogs was linear with respect to time for at least 30 min.

UV–Vis spectroscopy

Determination of P450 content

Purified P450terf was diluted in 0.1 M potassium phosphate buf-

fer, pH 7.4, and split between two cuvettes. The P450 content was

determined using the absorbance difference (D A450–490 nm) in the

difference spectrum of dithionite reduced P450 in the presence

of CO minus reduced P450 [23], using an Uvikon 930 spectropho-

tometer (Kontron Instruments).

Spectral interactions and binding titrationsPurified P450terf was diluted to 0.8 lM in 0.1 M potassium phos-

phate buffer, pH 7.4, and divided into two cuvettes. Terfenadine

was dissolved in a 1:1 DMF:water mixture and added at concentra-

tions varying from 0.1 to 100 lM. UV–Vis spectra (300–500 nm)

were recorded after stepwise additions of terfenadine, and the

difference spectra between samples with terfenadine and with

DMF:water alone were recorded [24]. Apparent dissociation con-

stants, K s, were determined by plotting the absorbance changes

DF390–418 nm calculated for each difference spectrum against the

Fig. 2. Terfenadine analogues used as P450terf substrates.

M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx 3


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concentration of terfenadine [S], and fitting the data to a hyperbola

according to Eq. (1), using Kaleidagraph software.

DAbs ¼DAbsmax Â ½S�

K s þ ½S�ð1Þ

Molecular biology methods

Genomic DNA extractionS. platensis genomic DNA extraction was performed according to

a general phenol/chloroform protocol, allowing DNA isolation with

a very high purity [25].

Sequencing of the gene encoding P450terf

The above purified genomic DNA from S. platensis was used as a

template for PCR using degenerate primers. The amplification of

the cytochrome P450 target sequence was carried using 1 lg geno-

mic DNA with 2.5 units of Kod polymerase and 50 pmol of a set of

degenerate primers. The sense primers 50 ATG TCC(G,T) GAG(A)

ATC ATC GAC(T) CTC(G,T,A)30 included the start codon (under-

lined), and two antisense primers were used: the first one, 50

G(A)CA G(A)AA G(A)TG A(G)AT G(T,A)CC G(A)TG G(C)CC GAA 30

,contained the codon corresponding to the cysteine ligand (under-

lined) from the P450 Cys pocket, and the second one, 5 0 TCA CCA

G(C,T,A)CG C(G,T,A)AC C(G)GG C(G)AG G(C)30, included the stop

codon (underlined). The amplification reactions were performed

according to the following scheme with a Thermal cycler MJ Mini

instrument (Bio-Rad, Marnes-la-Coquette): 60 s at 95 °C, 60 s at

62 °C, and 40 s at 72 °C for 30 cycles. After separation on an agarose

gel, the PCR products with the expected lengths were excised and

recovered using QIAquick by Qiagen (Courtabeouf, France). They

were sequenced on both strands and doubly read (Cogenics,

Meylan, France).

Nucleotide sequence accession number

The DNA and protein sequences have been deposited in theEMBL database as accession number FR717427 (01-11-2010).

Results

Oxidation of terfenadine by S. platensis whole cells and subcellular fractions

Cultures of S. platensis cells grown for 48 h were incubated with

terfenadine and then analyzed by HPLC–ESI-MS/MS. Cells culti-

vated in media with or without soybean peptones were able to oxi-

dize terfenadine into hydroxyterfenadine, and hydroxyterfenadine

into fexofenadine, as previously reported [15].

Next, the cytosol and membrane fractions from S. platensis cells

were prepared as previously described [16], and the ability of eachfraction to oxidize terfenadine in vitro was studied. In the presence

of NADPH or NADH alone, none of these fractions was able to

oxidize terfenadine into hydroxyterfenadine or fexofenadine. Then,

thereactions were performed in thepresence of an artificial electron

transfer chain, consisting of spinach ferredoxin (Fd) and NADPH:

ferredoxin oxidoreductase (FdR), which may provide S. platensisP450s with electrons in a possible reconstituted monooxygenase

[26].

In the presence of NADPH and the two electron transfer pro-

teins, the cytosol fraction was found to oxidize terfenadine into

hydroxyterfenadine, while the membrane fraction was unable to

catalyze this reaction under those conditions. It is noteworthy that

the cytosol fraction catalyzed the oxidation of terfenadine into

hydroxyterfenadine but was not able to catalyze the oxidation of hydroxyterfenadine into fexofenadine. The following experiments

were undertaken in order to purify the P450 present in the cytosol

fraction that is responsible for terfenadine hydroxylation. This en-

zyme will be referred to as P450terf in the further text.

Purification of P450terf

In order to purify P450terf , we developed a five-step purification

scheme allowing its isolation in an apparent homogeneous form.The results of a typical purification protocol are given in Table 1.

P450terf was purified by ammonium sulfate fractionation of the

cytosol proteins, followed by successive separations on Sephacryl

S-200, Uno Q, Phenyl Sepharose CL4-B and Superdex 200 columns.

With this protocol, P450terf was purified over-3000 fold to near

homogeneity, with a final 10% yield: 0.1 mg of purified protein

was obtained from 400 g (wet weight) of S. platensis cells contain-

ing 3200 mg of soluble proteins. Therefore, this P450 represented

approximately 0.003% of the total amount of the cytosolic proteins.

Purified P450terf showed a protein band on an SDS polyacrylamide

gel electrophoresis (Fig. 3), with an apparent molecular mass of

about 45 kDa. Purity of P450terf in the final fraction was also eval-

uated on the basis of the protein concentration according to the

Bradford method and of the D A(450–490 nm) in the difference spec-

trum. Assuming an extinction coefficient (De450À490) value of

91,000 MÀ1 cmÀ1, P450terf was found to be 90% pure.

Characterization of P450terf

UV–Vis spectroscopy

The UV–Vis absorption spectrum of purified P450terf was re-

corded between 350 and 700 nm. It showed features typical of a

heme-containing protein (Fig. S1 of Supplementary material). In

its resting state, P450terf exhibited a UV–Vis spectrum characteris-

tic of P450s Fe(III) in their low-spin hexacoordinate state, with a

Soret peak at 418 nm and a and b bands at 532 and 564 nm,

respectively. Upon reduction with dithionite and after bubbling

of CO, the Soret peak shifted to 450 nm as expected for a P450Fe(II)–CO complex [23] (Fig. 4). This complex was stable for several

minutes, and did not convert to a partially denatured P450 com-

plex, which absorbs around 420 nm.

Binding of terfenadine to P450terf

Addition of terfenadine to a solution of P450terf in phosphate

buffer pH 7.4 led to a blue shift of its Soret peak from 418 to

390 nm (Fig. 5). This shift is typical of the binding of a substrate

to P450 in a site very close to the heme, that leads to a transition

from the hexacoordinate state of native low-spin P450 Fe(III) to

its pentacoordinate high spin state, after loss of an H2O iron ligand

[24]. Titration of P450terf by increasing amounts of terfenadine and

plotting D A(390–418 nm) as a function of terfenadine concentration

led to a spectral dissociation constant, K s, of the P450terf –terfena-dine complex of 2.6 ± 0.4 lM. This value indicated that terfenadine

was recognized by P450terf with a relatively high affinity, compared

with K s values reported for other cytochrome P450–substrate com-

plexes [27].

Determination of the N-terminal aminoacid sequence of P450terf

In order to identify P450terf , the purified protein was transferred

to a PVDF membrane, and its N-terminal sequence was analyzed.

This study showed that the first 12 amino acid residues were

SEIIDLGAYGPD. A protein–protein BLAST (Basic Local Alignment

Search Tool) analysis showed significant identities with the

N-terminus of P450s from the CYP107L subfamily from other

Streptomyces sp. bacteria (asshown in Fig.6 that compares thecom-

plete sequence of P450terf with those of several members of theCYP107L subfamily). For instance, a level of 66% sequence identity



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wasfound with the N-terminal sequenceof CYP107L6 fromStrepto-myces sp.

Sequencing of the p450terf gene

A ClustalW multiple sequence alignment of the P450s of the

CYP107L subfamily showed that there was a strict conservation of

the sequence of the cysteine pocket-containing loop, ending in the

heme-binding cysteine residue (FGHGIHFC) (Fig. 6). Thus, several

oligonucleotides were designed on the basis of this conserved se-quence and of the determined N-terminal sequence of purified

P450terf . Some of the forward and reverse primers produced a PCR

product with the correct length, which was sequenced. The 1047-

bp sequence encoded a 349 amino-acid polypeptide which had

68% sequence identity to the Streptomyces venezuelae cytochrome

P450 CYP107L1,indicating that wehadcloned a fragment of thetar-

getgene.In order to sequencethe missing30 partof the P450terf gene,

we subsequently PCR-amplified S. platensis genomic DNA using a

second set of degenerate primers. Theantisense primers were based

upon the short 30 part conserved sequences, which contained the

LPV(I)RW30 conserved sequenceof theP450sfromthe CYP107L sub-

family. The PCR product with the correct length was sequenced. Its

associated EMBL accession number is FR717427.

Protein sequence analysisThe 1206-bp sequence encoded a 402 amino acid polypeptide

(Fig. S2 of Supplementary material), whose calculated molecular

mass (44 301 Da) was very close to the value obtained by SDS poly-

acrylamide gel electrophoresis (45 kDa). The encoded polypeptide

exhibited 65% sequence identity to CYP107L1, also named PikC,

the enzyme from S. venezuelae that catalyzes the hydroxylation of

several macrolides [28,29]. It also exhibited 47% sequence identity

to CYP107A1 (also called P450 EryF), from Saccharopolysporaerythraea, which catalyzes the hydroxylation of 6-deoxyerythrono-

lide B [30]. In a more general manner, it exhibited 56–65% identity

with P450s from the 107L subfamily reported so far (Table 2).

P450terf fingerprint analysis

Purified P450terf was digested by trypsin for identification bypeptide mass fingerprint (PMF) in mass spectrometry. The peptide

Table 1

Purification report of P450terf from Streptomyces platensis NRRL2364. Measurements were done at 28 °C, in 0.1 M potassium phosphate buffer, pH 7.4 containing 1 mM NADPH,

1 lM spinach FdR, 4 lM spinach Fd, 100 lM terfenadone and 0.1–200 lg protein fractions. One unit of enzyme was defined as the number of nmol of hydroxyterfenadone formed

per minute.

Purification step Total protein (mg) Specific activity (U m gÀ1) Total activity (U) Yield (%) Purification (Fold)

Cell-free extract 3200 0.2 640 100 1

Sephacryl S200 514 1.14 586 91 5.7

UnoQ 126 3.4 428 67 17

Phenyl Sepharose 0.3 328 98 15 1640Superdex S200 0.1 640 64 10 3200

1 2 3 4 5 6

250 -150 -

100 -

75 -

50 -

37 -

25 -

20 -

P450terf

Fig. 3. SDS–PAGE of the proteins after each purification steps of P450 terf . Lane 1,

proteins markers (kDa); lane 2, cell-free extract; lane 3, Sephacryl S-200; lane 4,

UnoQ; lane 5, Phenyl Sepharose; lane 6 , Superdex 75.

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

380 400 420 440 460 480 500

A b s o r b a n c e

d i f f e r e n c e

Wavelength (nm)

Fig. 4. Difference UV–Vis spectrumof the P450terf Fe(II)CO complex vs P450terf Fe(II)

(0.77 lM P450terf ).

0

0.02

0.04

0.06

0.08

0.1

360 380 400 420 440 460 480

Wavelength (nm)

A b s o r b a n c e

390 nm 418 nm

Fig. 5. UV–Vis study of the interaction of P450terf with terfenadine. 0.8 lM P450terf

in 0.1 M phosphate buffer pH 7.4; other spectra obtained after successive additions

of terfenadine (0.1, 0.9, 1.7, 2.5, 10.4, 18.2 and 96.2 lM).

M. Lombard et al. / Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx 5


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mass map of P450terf allowed a positive identification based on 12

tryptic peptides mass values that matched the protein encoded by

the p450terf gene previously sequenced, with a total ion score of 477, and a sequence coverage of 35% (Fig. S3 of Supplementary

material).

Catalytic activity of P450terf

Oxidation of terfenadine 1 and its analogues, terfenadone 2,

ebastine 3, that is an isomer of terfenadone, and compound 4,

which derives from terfenadone by the loss of the terminal

Ph2C–OH moiety (Fig. 2), by purified P450terf in the presence of

NADPH, spinach ferredoxin (Fd), and spinach ferredoxin:NADP(+)

oxidoreductase (FdR), was followed by HPLC–ESI-MS/MS.

Oxidation of terfenadine and its above mentioned analogues

was highly regioselective, as it always occurred on the methyl

group of their t -butyl substituent. Formation of the corresponding

primary alcohols was established by comparison of the HPLCretention times and mass spectra of these metabolites with those

of previously described authentic samples [21]. Formation of prod-

ucts arising from an oxidation of the amine function of the terfen-

adine analogues was not detected by HPLC–ESI-MS/MS.All these reactions required the presence of the two electron

transfer proteins, Fd and FdR, and NADPH, which was found to

be a better electron donor than NADH (data not shown).

The regioselectivity of the oxidation of terfenadine analogues by

P450terf is identical to that previously reported for human CYP2J2

[31–33], whereas it is quite different from that previously found

for human CYP3A4, which favored the oxidation of the amine func-

tion [3,34].

Steady-state kinetic parameters were determined for the

hydroxylation of terfenadone 2, ebastine 3 and compound 4 by

P450terf , by HPLC coupled to UV–Vis spectroscopy, which can quan-

tify the corresponding products, based on their Ar–C@O chromo-

phore (Fig. 2). Terfenadone and ebastine hydroxylations were

characterized by K m values at the lM level (2.3 ± 0.1 lM and3.8 ± 0.1lM, respectively) (Table 3), in agreement with the K s

Fig. 6. Clustal W2 multiple alignment of the amino acid sequence of P450terf with those of others members from the CYP107L subfamily. Lane 1, P450terf (this study); lane 2,

CYP107L1 (O87605) from Streptomyces venezuelæ (PikC), 65% identity; lane 3, CYP107L6 (BD133544) from Streptomyces sp., 63% identity; lane 4, CYP107L2 (Q82LM3) from

Streptomyces avermitilis, 60% identity; lane 5, CYP107L8 (Q6V1M0) from Streptomyces sp. HK803, 55% identity. Multiple sequence alignment was imported to Jalview for

display. Bold-inverted and gray-shaded cells indicate >50% amino acid identity or similarity among all the sequences, respectively. Underlined aminoacids correspond to the

primers used for amplification of the p450terf gene by degenerated primers-based PCR. The cysteine ligand of the heme is marked with an asterisk sign. Red boxes encircle

amino acids which are homologous residues in P450terf and PikC and which are important for substrate recognition.



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value that was measured for terfenadine binding to P450terf

(2.6 ± 0.4 lM). These P450terf -catalyzed hydroxylations were also

characterized by high kcat values for P450-dependent reactions

(30 ± 3 and 25 ± 2 minÀ1, respectively). Actually, these kcat values

are very similar to those previously reported for the hydroxylation

of the same substrates by human CYP2J2 (36 ± 2 and 35 ± 3 minÀ1,

respectively) [33].

Accurate measurement of the kinetic parameters of P450terf -cat-

alyzed hydroxylation of terfenadine by HPLC coupled to UV spec-

troscopy was not possible because of the much weaker UV

absorbance of terfenadine, which is due to the absence of an Ar-

C@O moietyin thismolecule. However,hydroxylationof terfenadinecould be measured when using a relatively high substrate concen-

tration(200 lM) for whichsufficientamountsof hydroxylatedprod-

uct are formed. Under those conditions, the rate of terfenadine

hydroxylation corresponded to 27 ± 3 nmol hydroxyterfenadine

formed/min/nmolP450terf . This value is comparableto theone found

for P450terf -catalyzed terfenadone hydroxylation under the same

conditions, i.e., 29.9 ± 1 nmol hydroxyterfenadone formed/min/

nmol P450terf .

As mentioned previously, P450terf also catalyzed the hydroxyl-

ation of a quite different and smaller molecule, compound 4, with

a slightly lower kcat (10 ± 1 minÀ1). However, the K m value found in

this case was much higher (75 ± 4 lM) indicating that the

Ph2C(OH) moiety is important for recognition by P450terf .

Discussion

In an effort to find the enzyme responsible for the hydroxylation

of terfenadine by S. platensis, we first showed that a cytosol fraction

from S. platensis catalyzed terfenadine hydroxylation in the pres-

ence of NADPH and an artificial electron transfer system made up

of spinach Fd and FdR. The requirement for these electron transfer

proteins in terfenadine hydroxylation is easily understandable if

one considers that most bacterial P450-dependent monooxygena-

ses are class I systems in which the P450 is associated with a ferre-

doxin and an FAD-containing NAD(P)H-dependent ferredoxin

reductase [26,35]. These first data suggested that terfenadine

hydroxylation by S. platensis was dependent on a cytosolic P450,

presumably associated with electron transfer proteins found in bac-

terial class I monooxygenases. During cell membranes disruption,

the monooxygenase complex is likely dissociated. However, spin-

ach Fd and FdR efficiently replaced the S. platensis electron transferproteins for terfenadine hydroxylation by the cytosolic fraction.

Purification and characterization of the cytosolic protein

responsible for terfenadine hydroxylation confirmed that this pro-

tein was a P450. This identification was based upon the following

characteristics:

(1) The UV–Vis difference spectrum of the protein reduced by

dithionite in the presence of CO (vs the reduced protein)

exhibited a peak at 450 nm, which is characteristic of the

Fe(II)–CO complex of heme-cysteinate proteins. Moreover,

the absolute visible spectrum of the protein showed bands

at 418, 532 and 564 nm that are usual for P450s Fe(III) in

their low-spin hexacoordinate state. Finally, after addition

of the terfenadine substrate, the 418 nm peak shifted to390 nm (Fig. 5). This blue shift is usually found upon sub-

strate binding to P450s, and corresponds to the loss of the

axial heme water ligand with formation of a high-spin pen-

tacoordinate P450 Fe(III)–substrate complex [24].

(2) The molecular mass of the protein was estimated to be

around 45 kDa from SDS gel electrophoresis, as expected

for a bacterial P450. P450s reported so far from the 107L

subfamily have molecular masses between 43 and 46 kDa

(Table 2).

(3) The N-terminal sequence of the protein was found to be

highly similar to those of P450s from the 107L subfamily

(Fig. 6).

(4) The protein efficiently catalyzed a chemically difficult reac-

tion, the hydroxylation of a non-activated C–H bond fromthe terminal methyl group of at least four substrates, terfen-

Table 2

Some characteristics of the members of the CYP107L subfamily.

CYP subfamily Streptomyces strain Accession number (UniProt) Gene names Protein length (aa) Substrates % Identity Refs.

107L (P450terf ) platensis FR717427a P450terf 402 Terfenadine 100 this article

107L1 venezuelae O87605 pikC 416 Narbomycine 65 [29]

107L pristinaespiralis B5HEW4 ssdg_03651 401 – 65 b

107L griseus B1VVC4 sgr_1279 391 – 65 b

107L sp ACT-1 D1WWL0 sact1draft_2200 391 – 65 b

107L9 peucetius Q70AR3 cyp0854 392 – 64 b

107L6 sp. BD133544a – 396 Staurosporine 63 [41]

107L roseosporus D6ACU3 ssgg_06001 399 – 62 b

107L14 sp. Mg1 B4V290 ssag_01909 410 – 62 b

107L griseus B3IX56 it1-107mnpr-3 399 – 61 b

107L sviceus B5I942 sseg_08177 405 – 61 b

107L2 avermitilis Q82LM3 cyp8 393 – 60 b

107L scabies C9ZGI2 scab_18241 396 – 60 b

107L14 virginiae A6YRR5 – 402 – 59 b

107L3 tubercidicus Q595T3 cypLA 415 – 58 b

107L4 tubercidicus Q595R7 cypLC 415 – 57 b

107L8 sp. HK-803 Q6V1M0 plmS2 399 Phoslactomycin 55 [45]

107L7 narbonensis Q8KRW9 nbmL 102c – – b

107AF platensis B6ZIR6 pldB 399 Pladienolide 48 [56]

– Unknown gene names and/or enzymatic activities.a From the EMBL database.b Data from Uniprot.c Truncated protein.

Table 3

Enzymatic constants for the oxidation of terfenadine analogues by P450terf . Mea-

surements were done at 28 °C in 0.2 mL Tris buffer 50 mM, pH 7.6, containing 2%

DMSO for substrates solubilization, 5 nM P450terf , 2 lM spinach Fd and 1 lM FdR.

Substrates K m (lM) kcat (minÀ1) kcat /K m (lMÀ1 minÀ1)

Terfenadone 2 2.3 ± 0.1 30 ± 3 13

Ebastine 3 3.8 ± 0.2 25 ± 2 6.5

Compound 4 75 ± 4 10 ± 1 0.13







adine, terfenadone, ebastine and compound 4 (Table 3). Such

difficult hydroxylations are most often catalyzed by P450-

dependent monooxygenases [36,37].

Sequencing of the amplified p450terf gene revealed a DNA se-

quence encoding a 402 amino acid polypeptide which corre-

sponded to the PMF data obtained with the tryptic digested

purified protein. Analysis of the P450terf aminoacid sequenceclearly showed that it belongs to the CYP107L subfamily. It exhib-

ited between 56% and 65% identity with those of the previously re-

ported members of the 107L subfamily (Table 2). The highest

identity (65%) was found with CYP107L1 from S. venezuelae.

The previously reported members of the CYP107L subfamily all

come from various Streptomyces strains (Table 2). The biological

functions of most of them are unknown so far. However, several

CYP107L appear to be involved in the biosynthesis of secondary

metabolites (Fig. 7). For example, CYP107L1 fromS. venezuelae, also

named PikC, is involved in the biosynthesis of macrolide antibiot-

ics, by performing the oxidative tailoring of their macrolactone

ring. It catalyzes the oxidation of YC-17 and narbomycin into

methymycin and pikromycin, respectively [29,38,39]. These oxida-

tions are hydroxylations of non-activated carbons, either on the

macrolactone ring or on a lateral alkyl chain. The three-dimen-

sional structures of the CYP107L1 complexes with YC-17 and nar-

bomycin have been determined by X-ray crystallography [40].

They revealed two modes of binding of the desosamine substituent

of these antibiotics in the active site of PikC, which may explain the

flexibility of the enzyme with respect to macrolactone ring sub-

strates. The D50, E85 and E94 residues are important for substrate

recognition. Interestingly, P450terf also involves acidic amino acid

residues in the corresponding positions (Fig. 6).

CYP107L6 from Streptomyces sp. has been implicated in the bio-

synthesis of 7-hydroxystaurosporine, by catalyzing the 7-hydrox-

ylation of staurosporine [41]. It is noteworthy that staurosporine

is a secondary metabolite of Streptomyces staurosporeus [42].

CYP107L7 from Streptomyces narbonensis, also called NbmL, may

be involved in the biosynthesis of narbomycin [43]. CYP107L8 fromStreptomyces sp. HK-803, also called PlmS2, is involved in the bio-

synthesis of the antibiotic phoslactomycin B [44], by catalyzing

the 18-hydoxylation of a precursor of phoslactomycin [45].

In a more general manner, Streptomyces filamentous bacteria

are able to produce a great variety of secondary metabolites, that

encompass about two-thirds of the natural products employed in

medicine, such as antibacterial, antiviral and antitumor com-

pounds, and appear as very important biocatalysis tools [46,47].

The genomes of Streptomyces bacteria encode about one third of

all known bacterial P450s, and approximately 600 CYPs genes have

been reported from Streptomyces species in all searchable

databases.

Coming back to S. platensis, whose genome has not been se-

quenced so far, this microorganism was found to produce diverse

biologically active polyketides, such as oxytetracyclin [48], dorri-

gocins [49,50], migrastatins[51], leustroducsin B [52], pladienolides

[53], spirotetronates [54], and platensimycin [55]. Platensimycin

selectively inhibits cellular lipid biosynthesis of broad spectrum

Gram-positive bacteria,such as methicillin-resistant Staphylococcusaureus, and pladienolide D, a new potent anti-tumoral macrolide

agent, has recently entered clinical trials [56]. S. platensis not only

A

OO

O

O O

OOH

N (CH3)2

OO

O

O O

OOH

N (CH3)2

OH

Narbomycine Pikromycine

B

N N

NH

O

O

OH3CNH CH3

H3C

N N

NH

OOH

O

OH3CNH CH3

H3C

Staurosporine 7 OH-staurosporine

C

OO

O

PO

OH

OH

OH

NH2

OH

OO

O

PO

OH

OH

OH

NH2

OH

OH

B-MLPnicymotcalsohPyxordyHB-MLPnicymotcalsohP

CYP107L1

(PikC)

CYP107L6

CYP107L8

(PlmS2)

Fig. 7. Examples of enzymatic activities previously reported for some P450s of the 107L subfamily from Streptomyces species.







produces useful secondary metabolites, but is also able to oxidize

xenobiotics like naphtoquinone derivatives valuable for pharma-

ceutical industry [57]. However, only few P450s from this strain

have been described so far; they all belong to the CYP107 family.

For instance, PldB (CYP107AF) is involved in the biosynthesis of

pladienolide B, by catalyzing the 6-hydroxylation of the macrolac-

tone ring [56] (Table 3). The three other described CYP107 from

S. platensis, CYP107Z8, CYP107Z10 and CYP107Z11, have all beenisolated on the basis of their ability to oxidize avermectin, a macro-

lactone with anthelminthic and insecticidal properties, into 400-oxo

avermectin [58].

P450s in general [59–61], and bacterial ones in particular

[62,63], can be versatile and powerful biocatalysts. Thus, could

P450s from Streptomyces species also be useful for the oxidation

of xenobiotics, in particular for the oxidative transformation of

drugs? There are some examples of such reactions [64]. This in-

cludes, besides the aforementioned oxidation of avermectin into

400-oxoavermectin by CYP107Z enzymes, the 16a-hydroxylation

of progesterone by CYP163A2 from Streptomyces roseochromogenes[65], the hydroxylation of compactin into pravastatin by P450sca

from Streptomyces carbophilus [66], the oxidation of warfarin by

P450s from Streptomyces rimosus [67], the O-dealkylation of 7-eth-

oxy-coumarin by the sulfonylurea herbicide-inducible CYP105A1

(P450SU1) and CYP105B1 (P450SU2) from Streptomyces griseolus[68] and the oxidation of some xenobiotics, including camphor,

coumarin, testosterone, warfarin, and various promutagenic chem-

icals, such as aromatic hydrocarbons and amines, by CYP105D1

(P450soy) from S. griseus [20,69–71]. In that regard, CYP105D1

can be viewed as the bacterial counterpart to mammalian CYP3A4,

because of its broad substrate specificity.

The results described in this article showed that S. platensisP450terf , a member of the CYP107L subfamily, is quite efficient at

hydroxylation of some terfenadine derivatives. It catalyzes the

hydroxylation of terfenadone and of the drug ebastine, with K mvalues at the low lM level and kcat values around 30 minÀ1

(Table 3). It also catalyzes the hydroxylation of a xenobiotic, com-

pound 4, whose structure markedly differs from that of terfena-done or ebastine, although the kcat /km value found for this

reaction is much lower (Table 3).

These results confirm that Streptomyces P450s are not only

interesting tools in the biotechnological production of secondary

metabolites, such as antibiotic or antitumor compounds, but also

in the oxidative transformation of drugs, which is of utility for drug

metabolites production and fine chemical synthesis, or degrada-

tion of chemical pollutants [72]. The possible endogenous and

exogenous substrates of P450terf , and the biological role(s) of this

cytochrome in S. platensis are currently under study.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.abb.2011.01.008.

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Pl it thi ti l i M L b d t l A h Bi h Bi h (2011) d i 10 1016/j bb 2011 01 008



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