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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: murielle.lombard@parisdescartes.fr(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
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y a b b i
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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.
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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  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
4 M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx
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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).
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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.
6 M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx
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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
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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.
8 M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx
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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.
References
[1] R.V. Smith, J.P. Rosazza, Biotechnol. Bioeng. 17 (1975) 785–814.[2] K. Simpson, B. Jarvis, Drugs 59 (2000) 301–321.[3] C.H. Yun, R.A. Okerholm, F.P. Guengerich, Drug Metab. Dispos. 21 (1993) 403–
409.[4] K.H. Ling, G.A. Leeson, S.D. Burmaster, R.H. Hook, M.K. Reith, L.K. Cheng, Drug
Metab. Dispos. 23 (1995) 631–636.[5] B.C. Jones, R. Hyland, M. Ackland, C.A. Tyman, D.A. Smith, Drug Metab. Dispos.
26 (1998) 875–882.[6] S. Matsumoto, Y. Yamazoe, Br. J. Clin. Pharmacol. 51 (2001) 133–142.[7] T. Hashizume, S. Imaoka, M. Mise, Y. Terauchi, T. Fujii, H. Miyazaki, T.
Kamataki, Y. Funae, J. Pharmacol. Exp. Ther. 300 (2002) 298–304.
[8] S. Parikh, P. Gagne, V. Miller, C. Crespi, K. Thummel, C. Patten, DrugMetab. Rev.35 (2003) 379.
[9] C.M. Pratt, J. Mason, T. Russell, R. Reynolds, R. Ahlbrandt, Am. J. Cardiol. 83(1999) 1451–1454.
[10] S.H. Kawai, R.J. Hambalek, G. Just, J. Org. Chem. 59 (1994) 2620–2622.[11] S. Patel, L. Waykole, O. Repic, K.M. Chen, Synth. Commun. 26 (1996) 4699–
4710.[12] Q.K. Fang, C.H. Senanayake, H.S. Wilkinson, S.A. Wald, H. Li, Tetrahedron Lett.
39 (1998) 2701–2704.[13] R. Azerad, J. Biton, I. Lacroix, Novel Method for Preparing Fexofenadine, WO
Patent 99/47693, Hoesht Marion Roussel, 1999.[14] C. Mazier, M. Jaouen, M.A. Sari, D. Buisson, Bioorg. Med. Chem. Lett. 14 (2004)
5423–5426.[15] C. Mazier, M. Lombard, M.A. Sari, D. Buisson, Biocatal. Biotransform. 25 (2007)
401–407.[16] A. El Ouarradi, M. Lombard, D. Buisson, J. Mol. Catal. B 67 (2010) 172–178.[17] F.S. Sariaslani, D.A. Kunz, Biochem. Biophys. Res. Commun. 141 (1986) 405–
410.[18] M.K. Trower, F.S. Sariaslani, D.P. Okeefe, J. Bacteriol. 171 (1989) 1781–1787.[19] D.R. Nelson, The cytochrome P450 Homepage, Hum. Genomics (2009) 59–65.[20] M. Taylor, D.C. Lamb, R. Cannell, M. Dawson, S.L. Kelly, Biochem. Biophys. Res.
Commun. 263 (1999) 838–842.[21] A. El Ouarradi, I. Salard-Arnaud, D. Buisson, Tetrahedron 64 (2008) 11738–
11744.[22] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254.[23] T. Omura, R. Sato, J . Biol. Chem. 239 (1964) 2370–2378.[24] C.R. Jefcoate, Methods Enzymol. 52 (1978) 258–279.[25] Y.K. Lee, H.W. Kim, C.L. Liu, H.K. Lee, J. Microbiol. Methods 52 (2003) 245–
250.[26] K.J. McLean, M. Sabri, K.R. Marshall, R.J. Lawson, D.G. Lewis, D. Clift, P.R.
Balding, A.J. Dunford, A.J. Warman, J.P. McVey, A.M. Quinn, M.J. Sutcliffe, N.S.Scrutton, A.W. Munro, Biochem. Soc. Trans. 33 (2005) 796–801.[27] P.R. Ortiz de Montellano, third ed., Cytochrome P450: Structure, Mechanism
and Biochemistry, Plenum Publisher, New York, 2005.[28] Y.Q. Xue, L.S. Zhao, H.W.Liu, D.H. Sherman, Proc. Natl. Acad. Sci. USA 95(1998)
12111–12116.[29] Y.Q. Xue, D. Wilson, L.S. Zhao, H.W. Liu, D.H. Sherman, Chem. Biol. 5 (1998)
661–667.[30] J.R. Cupp-Vickery, T.L. Poulos, Nat. Struct. Biol. 2 (1995) 144–153.[31] T. Hashizume, S. Imaoka, M. Mise, Y. Terauchi, T. Fujii, H. Miyazaki, T.
Kamataki, Y. Funae, J. Pharmacol. Exp. Ther. 300 (2002) 298–304.[32] P. Lafite, S. Dijols, D. Buisson, A.C. Macherey, D.C. Zeldin, P.M. Dansette, D.
Mansuy, Bioorg. Med. Chem. Lett. 16 (2006) 2777–2780.[33] P. Lafite, F. Andre, D.C. Zeldin, P.M. Dansette, D. Mansuy, Biochemistry-Us 46
(2007) 10237–10247.[34] T. Hashizume, M. Mise, Y. Terauchi, L.O.T. Fujii, H. Miyazaki, T. Inaba, Drug
Metab. Dispos. 26 (1998) 566–571.[35] A.W. Munro, J.G. Lindsay, Mol. Microbiol. 20 (1996) 1115–1125.[36] B. Meunier, S.P. de Visser, S. Shaik, Chem. Rev. 104 (2004) 3947–3980.
[37] P.R. Ortiz de Montellano, Chem. Rev. 110 (2010) 932–948.[38] M.C. Betlach, J.T. Kealey, M.C. Betlach, G.W. Ashley, R. McDaniel, Biochemistry
37 (1998) 14937–14942.[39] E.I. Graziani, D.E. Cane, M.C. Betlach, J.T. Kealey, R. McDaniel, Bioorg. Med.
Chem. Lett. 8 (1998) 3117–3120.[40] D.H. Sherman, S.Y. Li, L.V. Yermalitskaya, Y.C. Kim, J.A. Smith, M.R. Waterman,
L.M. Podust, J. Biol. Chem. 281 (2006) 26289–26297.[41] H. Mori, NewCytochrome P450, Patent JP2002058490, Kyowa Hakko Chemical
Co., Ltd., 2002.[42] A. Furusaki, N. Hashiba, T. Matsumoto, A. Hirano, Y. Iwai, S. Omura, Bull. Chem.
Soc. Jpn. 55 (1982) 3681–3685.[43] A.R. Butler, N. Bate, D.E. Kiehl, H.A. Kirst, E. Cundliffe, Nat. Biotechnol. 20
(2002) 713–716.[44] N. Palaniaappan, B.S. Kim, Y. Sekiyama, H. Osada, K.A. Reynolds, J. Biol. Chem.
278 (2003) 35552–35557.[45] M.S. Ghatge, K.A. Reynolds, J. Bacteriol. 187 (2005) 7970–7976.[46] M.G. Watve, R. Tickoo, M.M. Jog, B.D. Bhole, Arch. Microbiol. 176 (2001) 386–
390.[47] R.H. Baltz, Nat. Biotechnol. 24 (2006) 1533–1540.
[48] J.M. McGuire, Improvementsin or Relating to Terramycin, Brit. Patent 713,795,Eli Lilly and Co., 1954.
[49] J.E. Hochlowski, D.N. Whittern, P. Hill, J.B. Mcalpine, J. Antibiot. 47 (1994) 870–874.
[50] J.P. Karwowski, M. Jackson, G. Sunga, P. Sheldon, J.B. Poddig, W.L. Kohl, S.Kadam, J. Antibiot. 47 (1994) 862–869.
[51] E.J. Woo, C.M. Starks, J.R. Carney, R. Arslanian, L. Cadapan, S. Zavala, P. Licari, J.Antibiot. 55 (2002) 141–146.
[52] T. Kohama, T. Nakamura, T. Kinoshita, I. Kaneko, A. Shiraishi, J. Antibiot.(Tokyo) 46 (1993) 1512–1519.
[53] Y. Mizui, T. Sakai, M. Iwata, T. Uenaka, K. Okamoto, H. Shimizu, T. Yamori, K.Yoshimatsu, M. Asada, J. Antibiot. (Tokyo) 57 (2004) 188–196.
[54] M. Kang, B.D. Jones, A.L. Mandel, J.C. Hammons, A.G. DiPasquale, A.L.Rheingold, J.J. La Clair, M.D. Burkart, J. Org. Chem. 74 (2009) 9054–9061.
[55] J. Wang, S.M. Soisson, K. Young, W. Shoop, S. Kodali, A. Galgoci, R. Painter, G.Parthasarathy, Y.S. Tang, R. Cummings, S. Ha, K. Dorso, M. Motyl, H. Jayasuriya,
J. Ondeyka, K. Herath, C. Zhang, L. Hernandez, J. Allocco, A. Basilio, J.R. Tormo,O. Genilloud, F. Vicente, F. Pelaez, L. Colwell, S.H. Lee, B. Michael, T. Felcetto, C.
Gill, L.L. Silver, J.D. Hermes, K. Bartizal, J. Barrett, D. Schmatz, J.W. Becker, D.Cully, S.B. Singh, Nature 441 (2006) 358–361.
M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx 9
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[56] K. Machida, A. Arisawa, S. Takeda, T. Tsuchida, Y. Aritoku, M. Yoshida, H. Ikeda,Biosci. Biotechnol. Biochem. 72 (2008) 2946–2952.
[57] C. Fosse, L. Le Texier, S. Roy, M. Delaforge, S. Gregoire, M. Neuwels, R. Azerad,Appl. Microbiol. Biotechnol. 65 (2004) 446–456.
[58] I. Molnar, V. Jungmann, J. Stege, A. Trefzer, J.P. Pachlatko, Biochem. Soc. Trans.34 (2006) 1236–1240.
[59] F.P. Guengerich, Nat. Rev. Drug Disc. 1 (2002) 359–366.[60] R. Bernhardt, J. Biotechnol. 124 (2006) 128–145.[61] V.B. Urlacher, S. Eiben, Trends Biotechnol. 24 (2006) 324–330.[62] M.J. Cryle, J.E. Stok, J.J. De Voss, Aust. J. Chem. 56 (2003) 749–762.
[63] V. Urlacher, R.D. Schmid, Curr. Opin. Biotechnol. 13 (2002) 557–564.[64] D.C. Lamb, F.P. Guengerich, S.L. Kelly, M.R. Waterman, Expert Opin. Drug
Metab. 2 (2006) 27–40.
[65] J.R. Berrie, R.A.D. Williams, K.E. Smith, J. Steroid Biochem. 71 (1999) 153–165.[66] T. Matsuoka, S. Miyakoshi, K. Tanzawa, K. Nakahara, M. Hosobuchi, N.
Serizawa, Eur. J. Biochem. 184 (1989) 707–713.[67] R.J.P. Cannell, T. Rashid, I.M. Ismail, P.J. Sidebottom, A.R. Knaggs, P.S. Marshall,
Xenobiotica 27 (1997) 147–157.[68] H.A. Hussain, J.M. Ward, Appl. Environ. Microbiol. 69 (2003) 373–382.[69] F.S. Sariasiani, L.R. Mcgee, M.K. Trower, F.G. Kitson, Biochem. Biophys. Res.
Commun. 170 (1990) 456–461.[70] M. Ueno, M. Yamashita, M. Hashimoto, M. Hino, A. Fujie, J. Biosci. Bioeng. 100
(2005) 567–572.
[71] F.S. Sariaslani, R.G. Stahl, Biochem. Biophys. Res. Commun. 166 (1990) 743–749.
[72] F.P. Guengerich, Environ. Health Perspect. 103 (1995) 25–28.
10 M. Lombard et al./ Archives of Biochemistry and Biophysics xxx (2011) xxx–xxx
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