The fourth molybdenum containing enzyme mARC: Cloning and involvement in the activation of N-hydroxylated prodrugs

Article abstract of DOI:10.1021/jm8010417

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b ₅ and b₅ reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.

Full text of DOI:10.1021/jm8010417

J. Med. Chem. 2008, 51, 8173–8177
8173
The Fourth Molybdenum Containing Enzyme mARC: Cloning and Involvement in the
Activation of N-Hydroxylated Prodrugs
Sanja Gruenewald,† Bettina Wahl,‡ Florian Bittner,‡ Helen Hungeling,† Stephanie Kanzow,† Joscha Kotthaus,†
Ulrike Schwering,† Ralf R. Mendel,‡ and Bernd Clement*,†
Pharmaceutical Institute, Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-UniVersity of Kiel, Gutenbergstrasse 76,
D-24118 Kiel, Germany, Department of Plant Biology, Technical UniVersity of Braunschweig, Humboldtstrasse 1, D-38106 Braunschweig, Germany
ReceiVed August 19, 2008
The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated
compounds. Upon reconstitution with cytochrome b5 and b5 reductase, benzamidoxime, pentamidine, and
diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently
reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability
compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo
reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome
P450 enzymes.
Introduction
For 40 years, it was believed that only three molybdenum
containing enzymes are present in the human body. With the
recently purified protein from pig liver mitochondria named as
mitochondrial amidoxime reducing component, it became clear
that a fourth molybdenum containing enzyme exists which might
play a major role in drug metabolism though its physiological
relevance is not still clear.14
This mARC protein is unique as it forms the catalytic part
of a three-component enzyme complex. The N-reductive
complex located in the outer mitochondrial membrane with its
mARC protein represents the first eukaryotic molybdenum
enzyme with separate electron transport proteins. As the human
genome codes for two mARC-proteins, mARC1 and mARC2,
differences and similarities in function and substrate specificity
should be determined. In this study, the cloning, expression and
purification of mARC1 is described. Furthermore, the N-
reductive ability of the recombinant protein is demonstrated by
the reduction of different amidine and guanidine prodrugs.
Basic functional groups such as amidines and guanidines are
incorporated into various drug candidates and drugs. Because
of their basicity, these structures are protonated under physi-
ological conditions and are not absorbed from the gastrointestinal
tract. N-hydroxylation of amidines leads to compounds which
are less basic and not protonated under physiological conditions.
Because these properties result in sufficient oral absorption and
therefore in improved bioavailability the prodrug concept
amidoximes instead of amidines was developed.1 This prodrug
principle was applied to peptidomimetic inhibitors and antico-
agulants like melagatran2,3 and nearly 50 compounds in pre-
clinical and clinical development.4 Furthermore, amidines are
structural elements of trypanocidal and leishmanicidal com-
pounds such as pentamidine and diminazene.5,6 The guanidine
structure is also well represented in numerous drug classes
because its cation built by the protonation is often responsible
for the interaction with the negatively charged carboxylic
function of target molecules. The amidine and guanidine groups
imitate the structural part of arginine. The prodrug principle
developed for amidines can be applied to the guanidine
structures as well.7
This study should terminate a long debate on the enzymatic
basis of the reduction of these N-hydroxylated compounds. In
older studies, Kadlubar and Ziegler8,9 described a microsomal
and Bernheim10-12 a mitochondrial system consisting of cyto-
chrome b5 and NADH cytochrome b5 reductase and a third
unidentified component. The microsomal system was further
analyzed and a cytochrome P450a enzyme was identified in pig
liver microsomes as the third component.13 So far the ortholo-
gous human P450 isoenzyme has not been found. For the
mitochondrial enzyme system, the third component has further
been analyzed until it was purified from pig liver mitochondria.14
Results
Expression and Purification of Recombinant mARC1.
Expression of mARC1 in Escherichia coli yielded a His6-tagged
protein with a molecular mass of approximately 40 kDa (Figure
1A), which corresponded well to the calculated mass of 38.5
kDa. As in previous works, the activity of mARC was shown
to depend on molybdenum cofactor (Moco),14 the E. coli strain
TP1000 was chosen for recombinant expression of mARC1. E.
coli TP1000 is characterized by high accumulation of the
eukaryotic form of Moco, whereby sufficient saturation of
recombinant mARC1 with Moco in the required form should
be ensured. In fact, FormA-dephospho, the stable oxidation
product of both Moco and its metal-free precursor molybopterin
(MPT), could be identified on mARC1 (Figure 1B), indicating
that recombinant human mARC1 was purified in the cofactor-
loaded form. mARC1 was further analyzed for biologically
active Moco by use of the nit-1 reconstitution assay. This assay
is based on the transfer of Moco derived from an exogenous
source to the nitrate reductase apoprotein of the Moco-deficient
Neurospora crassa nit-1 mutant, whereby reconstitution of
NADPH-dependent nitrate reductase activity is achieved.15
When omitting supplementary molybdate from the reaction
* To whom correspondence should be addressed. Phone: +49-431-
8801126. Fax: +49-431-8801352. E-mail: bclement@pharmazie.uni-kiel.de.
†
Pharmaceutical Institute, Department of Pharmaceutical and Medicinal
Chemistry, Christian-Albrechts-University of Kiel.
‡
Department of Plant Biology, Technical University of Braunschweig.
a Abbreviations: BAO, benzamidoxime; P450, cytochrome P450; cyt,
cytochrome; DAO, diamidoxime; MAO, monoamidoxime; mARC, mito-
chondrial amidoxime reducing component; Moco, molybdenum cofactor;
MPT, molybdopterin; OMV, outer membrane vesicles; b5, cytochrome b5;
b5 reductase, NADH cytochrome b5 reductase.
10.1021/jm8010417 CCC: $40.75
2008 American Chemical Society
Published on Web 11/21/2008

8174 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Brief Articles
Table 1. N-Reduction of Benzamidoxime by the Reconstituted Systema
activity
composition of
incubation mixture
nmol·min-1
·
pmol·min-1
130.0 ( 42.8
mg-1 mARC1
complete system
1.30 ( 0.43b
complete system without BAO
without NADH
NDc
ND
without cyt b5
without b5 reductase
without mARC1
0.23 ( 0.00
0.20 ( 0.00
22.9 ( 0.5
20.0 ( 0.0
22.0 ( 0.3
only mARC1
ND
a A complete incubation mixture consisted of 200 pmol purified
microsomal cytochrome b5, 0.05 units of purified microsomal NADH
cytochrome b5 reductase (pig liver), 100 µg recombinant mARC1, 0.5 mM
benzamidoxime and 1 mM NADH in 150 µL of 100 mM phosphate buffer,
pH 6.0. For incubation, sample preparation and HPLC analysis see
Experimental Section or Supporting Information. Data are means ( SD of
four determinations. b Significant versus composition without cytochrome
b5 and without NADH cytochrome b5 reductase with p < 0.05 according
to Student′s t-test. c ND ) not detectable.
Figure 1. SDS-PAGE analysis of recombinant mARC1 and detection
of Moco/MPT bound to mARC1. (A) Recombinant human mARC1
(40 µg) purified by affinity chromatography was electrophoresed on a
10 % SDS-PA gel and stained with Coomassie Brilliant Blue (M )
molecular weight marker; masses are indicated in kDa). (B) Detection
of Moco/MPT bound to recombinant human mARC1. Protein-bound
Moco/MPT was converted into FormA-dephospho and detected via
HPLC by its specific fluorescence (excitation, 370 nm; emission, 450
nm). The dominant peak eluting at ∼6 min co-elutes with FormA-
dephospho prepared from the Mo-enzyme xanthine oxidase (XO), which
served as control.
Table 2. N-Reduction of N-Hydroxylated Compounds by the
Reconstituted Systema
specific activity
substrate
metabolite
nmol·min-1 ·mg-1 mARC1
benzamidoxime
benzamidine
pentamidine
pentamidine MAO
pentamidine
diminazene MAO
diminazene
1.30 ( 0.43
6.59 ( 0.35
0.56 ( 0.04
0.49 ( 0.12
4.31 ( 1.00
0.05 ( 0.02
0.48 ( 0.01
20.42 ( 4.56
15.94 ( 1.10
pentamidine MAO
pentamidine DAO
pentamidine DAO
diminazene DAO
diminazene DAO
N-hydroxymelagatran melagatran
guanoxabenz guanabenz
N-hydroxydebrisoquine debrisoquine
mixture, only active Moco but not Mo-free MPT can be detected
as only Moco is capable of reconstituting NADPH-nitrate
reductase activity. In the presence of molybdate however, MPT
is nonenzymatically converted to Moco, thereby enabling
reconstitution of NADPH-nitrate reductase activity as well.
mARC1 as purified after heterologous expression in E. coli
TP1000 yielded a specific NADPH-nitrate reductase activity of
2.7 ( 0.2 µmol nitrite/(mg·min) in the absence of molybdate,
while in the presence of molybdate, a specific activity of 3.2 (
0.3 µmol nitrite/(mg·min) was observed. These results indicate
that more than 80 % of the total cofactor bound to mARC1 is
represented by nit-1-active Moco, which was able to reconstitute
NADPH-nitrate reductase activity in the nit-1 extract.
a A complete incubation mixture consisted of 200 pmol purified
microsomal cytochrome b5, 0.05 units of purified microsomal NADH
cytochrome b5 reductase (pig liver), 100 µg mARC1, 0.5 mM substrate
and 1 mM NADH in 150 µl 100 mM phosphate buffer, pH 6.0. For
incubation, sample preparation and HPLC analysis see Experimental Section
or Supporting Information. Data are means ( SD of four determinations.
midoximes and their further reduction to amidines are demon-
strated (Table 2). Pentamidine monoamidoxime reduction to
pentamidine is catalyzed by mARC1 by an even higher
reduction rate than pentamidine diamidoxime to the correspond-
ing monoamidoxime and pentamidine. N-hydroxymelagatran is
reduced to melagatran (Table 2). The highest reduction rates
are achieved by in vitro assays with N-hydroxylated guanidines
(guanoxabenz reduction to guanabenz; N-hydroxydebrisoquine
to debrisoquine).
mARC1 is Capable of Reducing Benzamidoxime. Incuba-
tion with 0.5 mM benzamidoxime with the reconstituted enzyme
system containing NADH led to the formation of benzamidine.
The formation of benzamidine was determined by comparison
of the metabolite retention time with that of the synthetic
material using HPLC analysis. The relative rate of in vitro
reduction of benzamidoxime to benzamidine is shown in Table
1. The reaction was further specified by omitting single
components. Minor reduction rates could be found if no
cytochrome b5 or NADH cytochrome b5 reductase was added
to mARC1 (Table 1). The incubation mixture with only
cytochrome b5 and NADH cytochrome b5 reductase led to
benzamidine in a moderate extent. By omitting both electron
transport proteins, complete loss of mARC′s N-reductive activity
was observed. No N-reductive activity was detectable without
adding NADH (Table 1). The greatest conversion rates could
be detected if all three components were combined in presence
of NADH.
Reduction of N-Hydroxylated Compounds. The in vitro
reduction by mARC1 of all tested N-hydroxylated compounds
can be demonstrated (Table 2). Comparing different substrates,
there are significant variations concerning the N-reductive
activity of mARC1. All amidoxime prodrugs are reduced to their
amidines. Benzamidoxime as the model substrate for the
N-reductive activity is converted to benzamidine as also shown
in Table 1. The reduction of diamidoxime prodrugs to monoa-
Discussion
Many studies have been performed to identify the enzymes
responsible for the reduction of N-hydroxylated structures.9,13,16
The participation of cytochrome b5 and NADH cytochrome b5
reductase in these reductions has become clear in these studies.16
Whether a third component would be able to increase the
conversion rates or not has been questioned,17-19 but the
participation of a third component has been reported by
independent groups.8,10,13,16 While a P450 enzyme has repeat-
edly been postulated as the N-reductive component in pig liver
microsomes,13,20 the current study has shown that a molybdenum-
containing enzyme is responsible for the mitochondrial enzy-
matic activity.14 Additionally, the mitochondrial enzyme system
has turned out to be far more efficient than the microsomal
one.13,14 Therefore, it can be supposed that the reduction of
N-hydroxylated compounds in the human body is mainly

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8175
The in vitro system introduced in this study will be of great
benefit for further characterizations of the metabolism of many
N-hydroxylated compounds and for the development of novel
N-hydroxylated prodrugs, especially of amidines and guanidines.
Thus, the activation in the human body can be predicted in vitro.
The importance of conversions by molybdenum containing
enzymes will increase in the future because the conversion of
novel drugs by P450 enzymes will be tried to be avoided
because of potential interactions.22
Figure 2. Reduction of the model substrate benzamidoxime to
benzamidine by cytochrome b5, NADH cytochrome b5 reductase, and
mARC1.
performed by mitochondria as well. The specific reduction rates
of the recombinant mARC enzyme are comparable with the
human liver mitochondria.21 However, the third component has
so far only been purified from pig liver mitochondria. The
studies described in this work are for the first time based on
the human mitochondrial enzyme, which has been obtained as
a recombinant enzyme. The successfully demonstrated reduc-
tions show clearly that there is an enzyme system consisting of
three components that reduces N-hydroxylated compounds in
the human body. At this point, we identified the enzyme system
responsible for mitochondrial N-reductive activity.
The conversion rates indicate clearly that the highest conver-
sion rates are obtained in the presence of all three components.
This becomes especially obvious when the specific conversion
rates are referred to constant amounts of cytochrome b5 and
NADH cytochrome b5 reductase, respectively. The three com-
ponent system also shows the highest conversion rates if the
whole amount of protein in the incubation mixture is included.
The ideal ratio of cytochrome b5 and NADH cytochrome b5
reductase has been claimed to be 10:1.17 This ratio has been
used for the studies described. The conversion could be
increased by the addition of mARC to an incubation using the
Figure 3. Reduction of amidoxime prodrugs to amidines by cytochrome b5, NADH cytochrome b5 reductase, and mARC1.
Figure 4. Reduction of N-hydroxylated guanidines to guanidines by cytochrome b5, NADH cytochrome b5 reductase, and mARC1.

8176 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Brief Articles
proposed optimal ratio of cytochrome b5 and NADH cytochrome
b5 reductase. Although microsomal and not mitochondrial
cytochrome b5 and NADH cytochrome b5 reductase have been
used for the studies described in this paper, it is very unlikely
that this will influence the results. Outer mitochondrial mem-
brane and microsomal cytochrome b5 are products of different
genes but demonstrate a large degree of homology.23 In the case
of NADH cytochrome b5 reductase, the proteins are identical
and arise from the same gene.24
Finally, the results shown present a fourth human molybde-
num containing enzyme system in humans, which seems to be
an excellent catalyst for reducing N-hydroxylated compounds.
Whether this is also the physiological function has to be further
investigated.
The details show that mARC is significantly involved in the
activation of amidoxime prodrugs. The in vivo reduction of such
amidoxime prodrugs has been proven by several clinical studies,
especially with ximelagatran2 and sibrafiban.25 The simple
benzamidoxime (Figure 2) and N-hydroxymelagatran (Figure
3) have been used as model substances in this study. It can be
assumed that all other amidoxime prodrugs will be reduced in
a similar manner. There are about 50 amidoxime prodrugs,
which will ensure oral bioavailability of amidines in preclinical
and clinical development, respectively.4
The prodrugs of pentamidine and diminazene (Figure 3) were
used in this study as examples for orally available antitrypa-
nosomal and antileishmanial bisamidines.1 Besides derivatives
of pentamidine and diminazene developed in our laboratories,
other similar compounds like furamidine prodrugs26 are in
advanced clinical development. These amidoximes are methy-
lated at the oxygen. Those methoxy compounds are initially
dealkylated in order to expose the amidoxime. It can be assumed
that the in vivo reduction of N-hydroxylated derivatives of
furamidine is performed by the same three-component enzyme
system and not only by cytochrome b5 and NADH cytochrome
b5 reductase, as postulated.19
N-hydroxyguanidines, which are suited as prodrugs for
guanidines,7 are also converted by this enzyme system. In the
present study, guanoxabenz and N-hydroxydebrisoquine (Figure
4) have been used as model substances and the highly efficient
reduction has been shown. In this context, it can be speculated
that the physiologically occurring N-hydroxy-L-arginine27 is also
reduced by this enzyme system. This is the purpose of presently
ongoing research and might give further insight into a possible
physiological role of mARC.
In the present study, for the first time, an enzyme system
with a human recombinant third component has successfully
been reconstituted and its contribution to the activation of
amidoxime prodrugs has been demonstrated. It can serve as a
model system in order to predict the in vivo situation and also
to explain known reductions observed in vivo. The study also
demonstrates that the prodrug principle amidoximes instead of
amidines is not dependent on P450 enzymes. Thus, P450-
dependent interactions are avoided, which also explains why
those have not been observed for example in the case of
ximelagatran.2,3
the amidoxime prodrugs is done by human enzymes not related
to drug interactions.
Experimental Section
Materials. Benzamidoxime was synthesized from benzonitrile
and hydroxylamine as described.28 Pentamidine was obtained from
Sanofi Aventis (Frankfurt, Germany). The pentamidine diamidoxime
and monoamidoxime were synthesised in our laboratory as de-
scribed by Clement and Raether5 and Clement et al.29 Melagatran
and N-hydroxymelagatran were kindly supplied by AstraZeneca
(Mo¨lndal, Sweden). Diminazene and its amidoxime prodrugs were
synthesized as described.30 Debrisoquine sulfate was obtained from
Sigma (Taufkirchen, Germany). N-Hydroxydebrisoquine was syn-
thesized as described by Bailey and DeGrazia using dioxane instead
of N,N-dimethylformamide in the last reaction step.31 Guanabenz
acetate was kindly supplied by Wyeth-Pharma GmbH (Muenster,
Germany), guanoxabenz-HCl by Laboratoires Houde´ (Paris, France).
Acetonitrile, methanol, and acetic acid were purchased from J. T.
Baker (Deventer, Holland). All other chemicals were commercially
available and of analytical grade. Solvents used for HPLC were
all of HPLC grade purity.
Cloning of Human mARC1. Total RNA prepared from human
HepG2 cells by using the E.Z.N.A. Total RNA kit (Peqlab,
Erlangen, Germany) was reverse transcribed with AMV-reverse
transcriptase (Promega, Mannheim, Germany) and oligo-d(T) primer
according to standard procedures. A mARC1 cDNA was obtained
by subsequent polymerase chain reaction using mARC1-specific
primers mARC1_forward (5′-ATA TAT GGA TCC ATG GGC
GCC GCC GGC TCC TCC GCG-3′) and mARC1-reverse (5′-AAA
TTT AAG CTT TTA CTG GCC CAG CAG GTA CAC AGG-3′)
deduced from GenBank entry NM_022746. By this procedure, a
full-length open reading frame of 1011 base pairs was obtained
encoding for a protein of 337 amino acids. In addition, restriction
sites for BamHI and HindIII were introduced at the 5′- and the
3′-ends, respectively, which enabled cloning of the mARC1 cDNA
into the pQE80 expression plasmid (Qiagen, Hilden, Germany)
downstream from a sequence encoding six NH2-terminal histidine
residues. Correctness of the introduced cDNA was confirmed by
sequencing. We note that a reproducible polymorphism was
identified that resulted in substutition of lysine187 by methionine.
However, this polymorphism is also found in the databases, e.g.,
between protein accessions AAH10619 and EAW93291, and thus,
obviously resembles a naturally occuring polymorphism.
Expression and Purification of Recombinant mARC1. Routine
protein expression of mARC1 was performed in freshly transformed
E. coli TP1000 cells.32 Cells were grown aerobically in Luria Broth
medium in the presence of 100 µg/mL ampicillin at 22 °C to a
A600 ) 0.1 before induction with 15-30 µM isopropyl-ꢀ-D-
thiogalactopyranoside and addition of 1 mM sodium molybdate.
After induction, cells were grown for further 20 h at 22 °C. Cells
were harvested by centrifugation and stored at -70 °C until use.
Cell lysis was achieved by several passages through a French
pressure cell, followed by sonication for 5 min on ice. After
centrifugation, 6 × histidin-tagged protein was purified on a
nickel-nitrilotriacetic acid superflow matrix (Qiagen, Hilden,
Germany) under native conditions at 4 °C according to the
manufacturer’s manual. Eluted fractions were analyzed by SDS-
PAGE. Molybdenum binding pterin bound to the purified proteins
was detected and quantified by converting it to the stable oxidation
product FormA-dephospho, according to Johnson and Rajago-
palan.33 Oxidation, dephosphory-lation, QAE chromatography, and
high performance liquid chromatography (HPLC) analysis were
performed as described previously.34 The identity of Moco/MPT
prepared as FormA-dephospho from mARC1 was proven by
comparing its retention time with that of FormA-dephospho
prepared from bovine xanthine oxidase (Sigma, Taufkirchen,
Germany).
Amidines and guanidines are very often elements of new drug
candidates as the cations formed after protonation interact with
carboxylates of the target protein. However, these functional
groups have negative pharmacokinetic properties. Replacements
of amidines and guanidines result usually in a high loss of
affinity. The prodrug principle amidoximes instead of amidines
allows the maintenance of the amidine function and also oral
bioavailability. This paper demonstrates that the activation of
Assay for Determination of the Reduction Rate. Incubations
were carried out under aerobic conditions at 37°C in a shaking water
bath. If not otherwise stated, standard incubation mixtures of the
reconstituted system contained 100 µg mARC1, 0.05 U NADH

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8177
tion and partial purification of the rat and human enzyme systems
active in the reduction of N-hydroxymelagatran and benzamidoxime.
Drug Metab. Dispos. 2005, 33, 570–578.
cytochrome b5 reductase, 200 pmol cytochrome b5, 0.5 mM of the
N-hydroxylated substrate, and 1.0 mM NADH in a total volume of
150 µL of 100 mM potassium phosphate buffer pH 6.0. After
preincubation for 3 min at 37°C, the reaction was started by NADH
and terminated after 15 or 20 min by adding aliquots of methanol.
The precipitated proteins were sedimented by centrifugation and
the supernatant was analyzed by HPLC.
(17) Kurian, J. R.; Bajad, S. U.; Miller, J. L.; Chin, N. A.; Trepanier, L. A.
NADH cytochrome b5 reductase and cytochrome b5 catalyze the
microsomal reduction of xenobiotic hydroxylamines and amidoximes
in humans. J. Pharmacol. Exp. Ther. 2004, 311, 1171–1178.
(18) Kurian, J. R.; Chin, N. A.; Longlais, B. J.; Hayes, K. L.; Trepanier,
L. A. Reductive detoxification of arylhydroxylamine carcinogens by
human NADH cytochrome b5 reductase and cytochrome b5. Chem.
Res. Toxicol. 2006, 19, 1366–1373.
(19) Saulter, J. Y.; Kurian, J. R.; Trepanier, L. A.; Tidwell, R. R.; Bridges,
A. S.; Boykin, D. W.; Stephens, C. E.; Anbazhagan, M.; Hall, J. E.
Unusual dehydroxylation of antimicrobial amidoxime prodrugs by
cytochrome b5 and NADH cytochrome b5 reductase. Drug Metab.
Dispos. 2005, 33, 1886–1893.
(20) Clement, B.; Behrens, D.; Amschler, J.; Matschke, K.; Wolf, S.;
Havemeyer, A. Reduction of sulfamethoxazole and dapsone hydroxy-
lamines by a microsomal enzyme system purified from pig liver and
pig and human liver microsomes. Life Sci. 2005, 77, 205–219.
(21) Clement, B.; Mau, S.; Deters, S.; Havemeyer, A. Hepatic, extrahepatic,
microsomal, and mitochondrial activation of the N-hydroxylated
prodrugs benzamidoxime, guanoxabenz, and Ro 48-3656 ([[1-[(2s)-
2-[[4-[(hydroxyamino)iminomethyl]benzoyl]amino]-1-oxopropyl]-4-
piperid inyl]oxy]-acetic acid). Drug Metab. Dispos. 2005, 33, 1740–
1747.
(22) Torres, R. A.; Korzekwa, K. R.; McMasters, D. R.; Fandozzi, C. M.;
Jones, J. P. Use of density functional calculations to predict the
regioselectivity of drugs and molecules metabolized by aldehyde
oxidase. J. Med. Chem. 2007, 50, 4642–4647.
Acknowledgment. We thank Petra Ko¨ster for technical
assistance.
Supporting Information Available: Purification of cytochrome
b5 and NADH cytochrome b5 reductase, enzyme assays, instru-
mentation (HPLC data for all compounds), and references. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Clement, B. Reduction of N-hydroxylated compounds: amidoximes
(N-hydroxyamidines) as pro-drugs of amidines. Drug Metab. ReV.
2002, 34, 565–579.
(2) Gustafsson, D.; Nystrom, J.; Carlsson, S.; Bredberg, U.; Eriksson, U.;
Gyzander, E.; Elg, M.; Antonsson, T.; Hoffmann, K.; Ungell, A.;
Sorensen, H.; Nagard, S.; Abrahamsson, A.; Bylund, R. The direct
thrombin inhibitor melagatran and its oral prodrug H 376/95: intestinal
absorption properties, biochemical and pharmacodynamic effects.
Thromb. Res. 2001, 101, 171–181.
(3) Clement, B.; Lopian, K. Characterization of in vitro biotransformation
of new, orally active, direct thrombin inhibitor ximelagatran, an
amidoxime and ester prodrug. Drug Metab. Dispos. 2003, 31, 645–
651.
(23) Sergeev, G. V.; Gilep, A. A.; Estabrook, R. W.; Usanov, S. A.
Expression of outer mitochondrial membrane cytochrome b5 in
Escherichia coli purification of the recombinant protein and studies
of its interaction with electron-transfer partners. Biochemistry (Mos-
cow) 2006, 71, 790–799.
(24) Shirabe, K.; Landi, M. T.; Takeshita, M.; Uziel, G.; Fedrizzi, E.;
Borgese, N. A novel point mutation in a 3′ splice site of the NADH-
cytochrome b5 reductase gene results in immunologically undetectable
enzyme and impaired NADH-dependent ascorbate regeneration in
cultured fibroblasts of a patient with type II hereditary methemoglo-
binemia. Am. J. Hum. Genet. 1995, 57, 302–310.
(25) Wittke, B.; Mackie, I. J.; Machin, S. J.; Timm, U.; Zell, M.; Goggin,
T. Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single
ascending oral doses of sibrafiban, an oral platelet aggregation
inhibitor, in healthy male volunteers. Br. J. Clin. Pharmacol. 1999,
47, 521–530.
(26) Lanteri, C. A.; Trumpower, B. L.; Tidwell, R. R.; Meshnick, S. R.
DB75, a novel trypanocidal agent, disrupts mitochondrial function in
Saccharomyces cereVisiae. Antimicrob. Agents Chemother. 2004, 48,
3968–3974.
(27) Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.; Feldman,
P. L.; Wiseman, J. N-omega-Hydroxy-L-arginine is an intermediate
in the biosynthesis of nitric oxide from L-arginine. J. Biol. Chem. 1991,
266, 6259–6263.
(28) Kru¨ger, P. Ueber Abko¨mmlinge des Benzenylamidoxims. Ber. Dtsch.
Chem. Ges. 1885, 18, 1055–1060.
(29) Clement, B.; Bu¨renheide, A.; Rieckert, W.; Schwarz, J. Diacetyldia-
midoximeester of pentamidine, a prodrug for treatment of protozoal
diseases: synthesis, in vitro and in vivo biotransformation. ChemMed-
Chem 2006, 1, 1260–1267.
(30) Clement, B.; Immel, M.; Raether, W. Metabolic N-hydroxylation of
diminazene in vitro. Arzneimittelforschung 1992, 42, 1497–1504.
(31) Bailey, D. M.; DeGrazia, C. G. Hydroxyguanidines. A new class of
antihypertensive agents. J. Med. Chem. 1973, 16, 151–156.
(32) Palmer, T.; Santini, C. L.; IobbiNivol, C.; Eaves, D. J.; Boxer, D. H.;
Giordano, G. Involvement of the narJ and mob gene products in distinct
steps in the biosynthesis of the molybdoenzyme nitrate reductase in
Escherichia coli. Mol. Microbiol. 1996, 20, 875–884.
(4) Peterlin-Masic, L.; Cesar, J.; Zega, A. Metabolism-directed optimi-
sation of antithrombotics: the prodrug principle. Curr. Pharm. Des.
2006, 12, 73–91.
(5) Clement, B.; Raether, W. Amidoximes of pentamidine: synthesis,
trypanocidal and leishmanicidal activity. Arzneimittelforschung 1985,
35, 1009–1014.
(6) Bray, P. G.; Barrett, M. P.; Ward, S. A.; de Koning, H. P. Pentamidine
uptake and resistance in pathogenic protozoa: past, present and future.
Trends Parasitol. 2003, 19, 232–239.
(7) Clement, B.; Demesmaeker, M.; Linne, S. Microsomal catalyzed
N-hydroxylation of guanabenz and reduction of the N-hydroxylated
metabolite: characterization of the two reactions and genotoxic
potential of guanoxabenz. Chem. Res. Toxicol. 1996, 9, 682–688.
(8) Kadlubar, F. F.; McKee, E. M.; Ziegler, D. M. Reduced pyridine
nucleotide-dependent N-hydroxy amine oxidase and reductase activities
of hepatic microsomes. Arch. Biochem. Biophys. 1973, 156, 46–57.
(9) Kadlubar, F. F.; Ziegler, D. M. Properties of a NADH-dependent
N-hydroxy amine reductase isolated from pig liver microsomes. Arch.
Biochem. Biophys. 1974, 162, 83–92.
(10) Bernheim, M. L.; Hochstein, P. Reduction of hydroxylamine by rat
liver mitochondria. Arch. Biochem. Biophys. 1968, 124, 436–442.
(11) Bernheim, M. L. The hydroxylamine reductase of mitochondria. Arch.
Biochem. Biophys. 1969, 134, 408–413.
(12) Bernheim, M. L. The reduction of hydroxylamine and some aryl
hydroxamates by liver mitochondria from mammals and birds.
Enzymologia 1972, 43, 167–176.
(13) Clement, B.; Lomb, R.; Mo¨ller, W. Isolation and characterization of
the protein components of the liver microsomal O2-insensitive NADH-
benzamidoxime reductase. J. Biol. Chem. 1997, 272, 19615–19620.
(14) Havemeyer, A.; Bittner, F.; Wollers, S.; Mendel, R.; Kunze, T.;
Clement, B. Identification of the missing component in the mitochon-
drial benzamidoxime prodrug-converting system as a novel molyb-
denum enzyme. J. Biol. Chem. 2006, 281, 34796–34802.
(15) Nason, A.; Lee, K. Y.; Pan, S. S.; Ketchum, P. A.; Lamberti, A.;
DeVries, J. In vitro formation of assimilatory reduced nicotinamide
adenine dinucleotide phosphate: nitrate reductase from a Neurospora
mutant and a component of molybdenum enzymes. Proc. Natl. Acad.
Sci. U.S.A 1971, 68, 3242–3246.
(33) Johnson, J. L.; Rajagopalan, K. V. Structural and metabolic relationship
between the molybdenum cofactor and urothione. Proc. Natl. Acad.
Sci. U.S.A 1982, 79, 6856–6860.
(34) Schwarz, G.; Boxer, D. H.; Mendel, R. R. Molybdenum cofactor
biosynthesis. The plant protein Cnx1 binds molybdopterin with high
affinity. J. Biol. Chem. 1997, 272, 26811–26814.
(16) Andersson, S.; Hofmann, Y.; Nordling, A.; Li, X. Q.; Nivelius, S.;
Andersson, T. B.; Ingelman-Sundberg, M.; Johansson, I. Characteriza-
JM8010417