Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians

Article abstract of DOI:10.1124/dmd.113.055202

Human molybdenum-containing enzyme mitochondrial amidoxime reducing component (mARC), cytochrome b5 type B, and NADH cytochrome b5 reductase form an N-reductive enzyme system that is capable of reducing N-hydroxylated compounds. Genetic variations are known, but their functional relevance is unclear. Our study aimed to investigate the incidence of nonsynonymous single nucleotide polymorphisms (SNPs) in the mARC genes in healthy Caucasian volunteers, to determine saturation of the protein variants with molybdenum cofactor (Moco), and to characterize the kinetic behavior of the protein variants by in vitro biotransformation studies. Genotype frequencies of six SNPs in the mARC genes (c. 493A>G, c. 560T>A, c. 736T>A, and c. 739G>C in MARC1; c. 730G>A and c. 735T>G in MARC2) were determined by pyrosequencing in a cohort of 340 healthy Caucasians. Protein variants were expressed in Escherichia coli. Saturation with Moco was determined by measurement of molybdenum by inductively coupled mass spectrometry. Steady state assays were performed with benzamidoxime. The six variants were of low frequency in this Caucasian population. Only one homozygous variant (c.493A; MARC1) was detected. All protein variants were able to bind Moco. Steady state assays showed statistically significant decreases of catalytic efficiency values for the mARC-2 wild type compared with the mARC-1 wild type (P < 0.05) and for two mARC-2 variants compared with the mARC-2 wild type (G244S, P < 0.05; C245W, P < 0.05). After simultaneous substitution of more than two amino acids in the mARC-1 protein, N-reductive activity was decreased 5-fold. One homozygous variant of MARC1 was detected in our sample. The encoded protein variant (A165T) showed no different kinetic parameters in the N-reduction of benzamidoxime. Copyright

Full text of DOI:10.1124/dmd.113.055202

1521-009X/42/4/718–725$25.00
http://dx.doi.org/10.1124/dmd.113.055202
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:718–725, April 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Functional Characterization of Protein Variants Encoded by
Nonsynonymous Single Nucleotide Polymorphisms in MARC1
and MARC2 in Healthy Caucasians
Gudrun Ott, Debora Reichmann, Cornelia Boerger, Ingolf Cascorbi, Florian Bittner,
Ralf-Rainer Mendel, Thomas Kunze, Bernd Clement, and Antje Havemeyer
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Kiel,
Germany (G.O., T.K., B.C., A.H.); Department of Plant Biology, Technical University of Braunschweig, Braunschweig, Germany
(D.R., F.B., R.-R.M.); and Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein, Campus Kiel,
Kiel, Germany (C.B., I.C.)
Received September 27, 2013; accepted January 14, 2014
ABSTRACT
Human molybdenum-containing enzyme mitochondrial amidoxime
reducing component (mARC), cytochrome b5 type B, and NADH
coli. Saturation with Moco was determined by measurement of
molybdenum by inductively coupled mass spectrometry. Steady state
cytochrome b5 reductase form an N-reductive enzyme system that is assays were performed with benzamidoxime. The six variants were of
capable of reducing N-hydroxylated compounds. Genetic variations low frequency in this Caucasian population. Only one homozygous
are known, but their functional relevance is unclear. Our study aimed variant (c.493A; MARC1) was detected. All protein variants were able
to investigate the incidence of nonsynonymous single nucleotide to bind Moco. Steady state assays showed statistically significant
polymorphisms (SNPs) in the mARC genes in healthy Caucasian
volunteers, to determine saturation of the protein variants with molyb- compared with the mARC-1 wild type (P < 0.05) and for two mARC-2
denum cofactor (Moco), and to characterize the kinetic behavior of the variants compared with the mARC-2 wild type (G244S, P < 0.05;
protein variants by in vitro biotransformation studies. Genotype fre- C245W, P < 0.05). After simultaneous substitution of more than two
decreases of catalytic efficiency values for the mARC-2 wild type
quencies of six SNPs in the mARC genes (c.493A>G, c.560T>A,
c.736T>A, and c.739G>C in MARC1; c.730G>A and c.735T>G in
MARC2) were determined by pyrosequencing in a cohort of 340
amino acids in the mARC-1 protein, N-reductive activity was de-
creased 5-fold. One homozygous variant of MARC1 was detected in
our sample. The encoded protein variant (A165T) showed no different
healthy Caucasians. Protein variants were expressed in Escherichia kinetic parameters in the N-reduction of benzamidoxime.
Introduction
molybdenum cofactor (Moco) and serves as a prosthetic group of
mARC. Eukaryotic molybdenum enzymes are classified by the co-
ordination chemistry of the molybdenum atom of Moco in two groups:
the xanthine oxidoreductase family and the sulfite oxidase family. mARC
proteins are regarded as members of the sulfite oxidase family (Chamizo-
Ampudia et al., 2011; Havemeyer et al., 2011; Rajapakshe et al., 2011;
Mendel and Kruse, 2012).
The subcellular localization of mARC proteins is not fully defined
(Hille et al., 2011). Previous studies in tissues of different species
showed that at least one isoform of mARC was detectable in
mitochondria (Da Cruz et al., 2003; Havemeyer et al., 2006; Wahl
et al., 2010; Havemeyer et al., 2011; Krompholz et al., 2012; Neve
et al., 2012). mARC-2 is also localized in peroxisomes (Islinger et al.,
2007; Wiese et al., 2007). Studies with human cell lines revealed that
mARC-1 is associated with the outer mitochondrial membrane with an
Mitochondrial amidoxime reducing component (mARC) was dis-
covered in 2006, and is the fourth molybdenum-containing enzyme in
humans (Havemeyer et al., 2006). There are two known isoforms of
mARC: mARC-1 and mARC-2. These isoforms are encoded by two
genes (MARC1, NM_022746.3; and MARC2, NM_017898.3), which
are located on chromosome 1 (1q41) in a tandem arrangement. Their
sequences show 66% identity and 80% similarity (Wahl et al., 2010).
The mARC proteins contain a mitochondrial targeting signal
sequence, a predicted b-barrel domain, and a molybdenum cofactor
sulfurase C-terminal domain- containing (MOSC) domain (Anantharaman
and Aravind, 2002). The MOSC domain is characterized by a con-
served cysteine in positions 273 and 272 of mARC-1 and mARC-2,
respectively (Anantharaman and Aravind, 2002; Hille et al., 2011).
mARC proteins contain molybdenum, which requires coordination
by a pyranopterin to gain biologic activity. This complex is named
N
(in)-C(out) membrane orientation. The catalytic domain is localized at
the C-terminus and exposed to the cytosol (Klein et al., 2012).
Molybdenum-containing enzymes in general show redox activity
(Hille et al., 2011). mARC-1 and mARC-2 exert N-reductive activity
together with cytochrome b5 type B (CYB5B, NM_030579.2) and
NADH cytochrome b5 reductase (CYB5R3, NM_007326.4) in the
This research was supported by the Deutsche Forschungsgemeinschaft [Grants
Cl-56/9-1 and ME-1266/24-1].
dx.doi.org/10.1124/dmd.113.055202.
ABBREVIATIONS: ANOVA, analysis of variance; CYB5B, cytochrome b5 type B (outer mitochondrial membrane); CYB5R3, cytochrome b5
reductase 3; HPLC, high-performance liquid chromatography; ICP-MS, inductively coupled mass spectrometry; mARC, mitochondrial amidoxime
reducing component; Moco, molybdenum cofactor; SNP, single nucleotide polymorphism; WT, wild-type.
718

SNPs in mARC
719
DNA Isolation. Whole genomic DNA was isolated from venous blood
samples by using the QIAamp DNA Blood Mini Kit (Qiagen, Hildesheim,
Germany).
presence of NADH (Havemeyer et al., 2006). Binding of Moco is
essential for the N-reductive activity of mARC. Wild-type (WT)
proteins of mARC-1 and mARC-2 expressed in the Escherichia coli
strains RK5204 and RK5206 do not contain Moco; consequently,
N-reductive activity is absent after reconstitution in the in vitro assay
together with cytochrome b5 type B and NADH cytochrome b5
reductase (Wahl et al., 2010).
The physiologic role of mARC and the N-reductive enzyme system
is still not known (Havemeyer et al., 2011). The mARC-containing
N-reductive enzyme system catalyzes the reduction of N-hydroxylated
substrates (Havemeyer et al., 2011). A putatively physiologic substrate
is the nitric oxide precursor Nv-hydroxy-L-arginine, which is involved in
the complex regulation of nitric oxide biosynthesis (Kotthaus et al.,
2011). Other putative substrates are N-hydroxylated base analogs. Their
reduction indicates a role of mARC in detoxification pathways (Wahl
et al., 2010; Krompholz et al., 2012). Moreover, amidoximes (N-
hydroxyamidines) can be used as prodrugs of amidines by lowering the
basicity of the amidines and thus preventing a positive charge and low
permeability. In vivo they are activated by reduction to amidines by the
mARC-containing N-reductive enzyme (Clement, 2002). Drugs carry-
ing this prodrug principle include ximelagatran as well as several drug
candidates in development (Clement and Lopian, 2003; Peterlin-Masic
et al., 2006; Froriep et al., 2013). Benzamidoxime is used as a model
compound for this prodrug principle (Clement, 2002).
Genotyping. National Center for Biotechnology Information dbSNP release
builds 130–133 were reviewed for SNPs in human MARC1 and MARC2. SNPs
were selected for genotyping according to the following criteria: nonsynonymous
SNPs, localization in the open reading frame for the N-truncated soluble
recombinant proteins, and notified SNPs not present in Caucasians only in
conjunction with a SNP, which fulfilled the above-mentioned criteria.
The following SNPs were determined by pyrosequencing on a PSQ 96HS
platform (Biotage AB, Uppsala, Sweden): c.493A.G (T165A, rs2642438),
c.560T.A (M187K, rs17850677), c.736T.A (C246S, rs3738178), and
c.739G.C (D247H, rs72470601) for MARC1; and c.730G.A (G244S,
rs3795535) and c.735T.G (C245W, rs76664695) for MARC2.
Primers were designed by Pyrosequencing Assay Design software (version
1.0; Biotage AB), and primer sequences are listed in Table 1. PCR was carried
out in a thermocycler in a reaction volume of 25 ml containing 2.5 ml 10 Â PCR
Rxn buffer - MgCl2 (Invitrogen, Darmstadt, Germany), 1.25 ml MgCl2 (50
mM; Invitrogen), 2.5 ml dNTP (2.5 mM; Biozym, Hessisch Oldendorf,
Germany), 0.5 ml primer F (10 mM; Sigma-Aldrich, Steinheim, Germany),
0.5 ml primer R (10 mM; Sigma-Aldrich), 0.15 ml Taq DNA Polymerase
(Invitrogen), 1 ml genomic DNA, and ultra-pure water (Invitrogen).
Thermocycler (Gene Amp PCR System 9700, version 3.08; Applied Bio-
systems, Darmstadt, Germany) conditions consisted of initial denaturation for
2 minutes at 95°C followed by 50 cycles of 30 seconds at 95°C, 30 seconds at
65°C (c.493A.G, c.560T.A, c.730G.A, c.735T.G) and 63°C (c.736T.A,
c.739G.C), respectively, and 30 seconds at 72°C, terminal elongation for
7 minutes at 72°C, and a final hold at 14°C. Agarose gel electrophoresis (7 ml
PCR product, 2% agarose gel, 120 V, 30 minutes) was performed to check the
length of the DNA fragment against a DNA ladder (GeneRuler 100bp DNA
Ladder; Fermentas, Darmstadt, Germany). Pyrosequencing followed a standard
protocol as essentially published previously (Haenisch et al., 2008).
Construction of Expression Vectors. Expression vectors were constructed as
Single nucleotide polymorphisms (SNPs) are known in both mARC
genes. At the beginning of our studies, six nonsynonymous SNPs in
MARC1 and two in MARC2, localized in the open reading frame for
the N-truncated soluble recombinant proteins, were registered in the
National Center for Biotechnology Information Single Nucleotide
Polymorphism database (dbSNP, release build 133; http://www.ncbi.
nlm.nih.gov/snp/). There are currently 41 nonsynonymous SNPs in previously described (Wahl et al., 2010). Variants c.286G.C, c.493G.A,
c.560T.A, c.736T.A, c.739G.C, and c.804G.A of MARC1 and c.730G.A
and c.735T.G of MARC2 were generated by PCR mutagenesis using primers
carrying the desired mutation. Variants leading to multiple amino acid changes in
mARC-1 were also generated by PCR mutagenesis. The six amino acid changes
were introduced in six steps using primers carrying the desired mutation. The order
of the mutations was c.493G.A followed by c.560T.A, c.286G.C, c.736T.A,
c.739G.C, and c.804G.A in MARC1. Accuracy of PCR mutagenesis was
confirmed by DNA sequencing (GATC, Konstanz, Germany).
Expression and Purification of Recombinant Proteins. Standard expres-
sion of the mARC proteins and variants was performed in freshly transformed
E. coli TP 1000 cells. Cells were supplemented with 1 mM sodium molybdate.
Expression of cytochrome b5 type B was performed in E. coli DL41. Cells were
supplemented with 1 mM aminolevulinic acid to support heme synthesis.
Expression of NADH cytochrome b5 reductase was performed in E. coli DL41.
A detailed description of expression and purification was previously published
(Wahl et al., 2010; Kotthaus et al., 2011).
Determination of Protein Concentrations. Concentrations of all recombi-
nant proteins were determined by a BCA assay after precipitating proteins
using the Compat-Able Protein Assay Preparation Reagent Set (Thermo
Scientific, Waltham, MA) and Pierce BCA Protein Assay Kits (Thermo
Scientific). All samples were measured at 562 nm (Cary 50; Varian Inc.,
Belrose, Australia). Measurement of each duplicate was repeated twice.
Determination of Cofactor Contents of the Recombinant Human
Proteins. Molybdenum content of the mARC proteins was measured by
inductively coupled plasma mass spectrometry (ICP-MS) as described earlier
(Kotthaus et al., 2011). The analytical error as determined from replicates and
certified reference materials was better than 3% RSD (relative standard
deviation; 1s). mARC proteins were eluted in elution buffer (50 mM sodium
phosphate, pH 8.0, containing 300 mM sodium chloride, 250 mM imidazole,
and 10% glycerol). Molybdenum content of mARC proteins was determined
after exchanging the buffer of the mARC proteins to exclude contamination
with sodium molybdate that was added for expression of the proteins. Initially,
molybdenum content of the proteins was measured with and without ex-
changing the buffer to 50 mM potassium phosphate, pH 7.4, containing
MARC1 and 27 in MARC2 (dbSNP build 138). To the best of our
knowledge, no studies have been published to date demonstrating
whether the resulting amino acid changes have any functional con-
sequences for the variant protein.
This study aimed to investigate the prevalence of nonsynonymous
SNPs in both mARC genes in healthy Caucasians, to determine
saturation with Moco of recombinant expressed human mARC pro-
teins (WT and variants) by measurement of molybdenum content, and to
characterize the enzyme kinetics of genetic mARC variants by in vitro
biotransformation studies using the model substrate benzamidoxime
(Fig. 1).
Materials and Methods
Subjects. Genotype and allele frequencies of four SNPs in MARC1 and two
SNPs in MARC2 were determined in DNA samples from a cohort of 340
nonrelated, healthy Caucasian volunteers of German origin (110 men and 230
women; mean age 33.3 years; range, 26–67 years). All subjects gave their
written informed consent and this study was approved by the Ethics Committee
of the Medical Faculty of Christian-Albrechts-University of Kiel.
Fig. 1. Reduction of benzamidoxime by the mARC-containing N-reductive enzyme
system. Benzamidoxime is reduced to benzamidine by mARC, cytochrome b5 type
B (Cyt b5), and NADH cytochrome b5 reductase (Cyt b5 R) in the presence of
NADH.

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Ott et al.
TABLE 1
Primers used for genotyping of four SNPs in MARC1 and two SNPs in MARC2
Fragment Length of
PCR Product
SNP
Primer Sequence
Tm
°C
bp
MARC1
c.493A.G
F: 59-Bio-AGTGCACGGCCTGGAGATAGAG
R: 59-TAGGGCTGTGACTTCAGGAAGCT
S: 59-CTGGTTATCCACTGG
F: 59-Bio-GCCCAGTGGATAACCAGCTTC
R: 59-TGGGTCGGAACAAGTCTGCTATT
S: 59-GGACGTCTCGGTCGC
F: 59-Bio-CTCAACTCCAGGCTAGAGAAGAA
R: 59-GATCCAAAGGGGCATAGTGTTAC
S: 59-TGTTACCTCTGCATAGACA
74.0
72.6
51.7
72.0
73.0
59.2
68.1
69.7
51.7
84
c.560T.A
109
107
c.736T.A
c.739G.C
MARC2
c.730G.A
F: 59-AGATGCCTCCCTGGTAGATTTGAA
R: 59-Bio-CTTACCTCCTCAAAAGCATCACAG
S: 59-GCCAAATATTGTGGTGAC
F: 59-Bio-TGCCTCCCTGGTAGATTTGAATA
R: 59-AAAAGCAGTGGGTCGCTTACCTC
S: 59-TACCTCCTCAAAAGCAT
73.1
70.3
53.9
70.8
73.7
50.4
108
120
c.730G.A
c.735T.G
Bio, biotin; F, forward primer used for PCR; R, reverse primer used for PCR; S, sequencing primer used for pyrosequencing; Tm,
melting temperature.
20% (m/v) glycerol, 0.1 mM dithiothreitol, and 1 mM sodium EDTA (Roth,
Karlsruhe, Germany). After ensuring that there was no difference between these
two methods of sample preparation, proteins were measured without exchanging
were separated by centrifugation (5 minutes, 10,000 rpm) and the amount of
product in the supernatant was determined by high-performance liquid
chromatography (HPLC). Reduction followed Michaelis–Menten kinetics.
the buffer. Measurement of each duplicate was repeated three times. Molybdenum Data were initially checked for substrate inhibition by using Lineweaver–Burk
saturation was calculated from molybdenum content and calculated molecular
masses of the mARC proteins.
Cytochrome b5 type B contains an N-terminal heme binding domain carrying
plots. Kinetic parameters Vmax and Km were determined through Michaelis–
Menten plots using SigmaPlot 11.0 (Systat Software, Chicago, IL). In cases of
substrate inhibition, kinetic parameters were calculated from data of the linear
1 mol heme/mol protein (Parthasarathy et al., 2011). Heme content was determined range of the Lineweaver–Burk plots. Each lot of mARC protein was incubated
by measurement of difference spectra according to Estabrook and Werringloer three times in duplicate and measured twice by HPLC. Incubations of multiple
(1978). Cytochrome b5 type B was reduced in the presence of NADH cytochrome
b5 reductase by NADH (Merck, Darmstadt, Germany). A wavelength scan from
400 to 500 nm was run (Cary 50). Absorption showed a maximum at 426 nm and
a minimum at 409 nm. The addition of NADH was repeated until the difference
variants of mARC contained duplicates that were measured twice.
Separation of Benzamidoxime from Benzamidine by HPLC. An HPLC
system was built comprising a Waters 1525 HPLC pump, a Waters 717
autosampler, and a Waters 2487 dual absorbance detector combined with
between maximum and minimum of absorption reached a maximum. Heme content Waters Breeze software version 3.30 (Waters Corporation, Milford, MA).
was calculated from the maximal difference between maximum and minimum HPLC analysis was performed on a LiChroCART 250-4 LiChrospher60 RP-
absorption using the molar extinction coefficient for the absorbance change at select B (5 mm) column and a LiChroCART 4-4 guard column (Merck,
426 nm 2 409 nm (« = 185 mM21 Â cm21). The assay was performed in dupli- Darmstadt, Germany). Elution was carried out isocratically with 10 mM
cate. Heme content of the lots of cytochrome b5 type B used in this work ranged 1-octylsulfonate sodium salt (Tokyo Chemical Industry, Tokyo, Japan) and
from 2.1 to 2.9 nmol heme/mg protein.
NADH cytochrome b5 reductase contains a flavoprotein domain carrying kept at 1 ml/min. Characteristic retention times were 7.9 6 0.1 minutes for
1 mol FAD/mol protein (Kensil and Strittmatter, 1986). FAD content was benzamidoxime and 26.7 6 0.1 minutes for benzamidine (Clement et al.,
17% (v/v) acetonitrile (Sigma-Aldrich). pH was not adjusted. The flow rate was
determined according to Whitby (1953). Samples were denatured at 100°C 2005).
for 10 minutes in the dark. Proteins were sedimented by centrifugation
Statistical Analysis. Data analysis of statistically significant differences was
(10 minutes, 10,000 rpm). Then, 200 ml supernatant of each sample was transferred
to a 96-well plate. Absorption was measured at 450 nm (Cary 50). Calculation of
FAD content was carried out using calibration in the range of 0.01–0.1 mmol FAD/
ml with an accuracy of the mean of 99.1% 6 3.1%. The assay was performed in
duplicate. FAD content of NADH cytochrome b5 reductase used in this work was
16 nmol FAD/mg protein.
Benzamidoxime. Benzamidoxime (N-hydroxy-benzene-carboximidamide)
was synthesized from benzonitrile and hydroxylamine as previously described
and was assayed as usual (Krüger, 1885).
Determination of Kinetic Parameters of the mARC Variants. Incubation
mixtures were adjusted on the cofactors of the three recombinant proteins. High
values of N-reductive activity were measured when high amounts of mARC
and cytochrome b5 type B and low amounts of NADH cytochrome b5 reductase
were added to incubation mixtures. mARC containing 120 pmol molybdenum,
cytochrome b5 type B containing 60 pmol heme, and NADH cytochrome b5
reductase containing 6 pmol FAD were incubated in steady state assays with
0.1–5 mM benzamidoxime in 20 mM 4-morpholineethanesulfonic acid, pH 6.0.
Incubations were performed under aerobic conditions at 37°C. After 3 minutes
of preincubation, the reaction was started by adding 1 mM NADH and was
performed with SigmaPlot 11.0 using a t test or one-way analysis of variance
(ANOVA) followed by a suitable post hoc test. P , 0.05 was considered
significant.
Results
Genotype Frequencies of SNPs in MARC1 and MARC2. The
SNPs c.493A.G, c.560T.A c.736T.A, and c.739G.C in MARC1
and c.730G.A and c.735T.G in MARC2 were determined by
pyrosequencing. It was possible to detect variant genotypes in 292 to
320 samples of the investigated cohort of 340 individuals. Frequency
distribution of alleles and genotypes is shown in Table 2.
For MARC1, the frequencies of all genotypes were in Hardy–
Weinberg equilibrium. The most frequently detected SNP was
c.493A.G; however, variant genotypes 493AG and GG were ob-
served more frequently than the reference genotype 493AA, which
had a frequency of only 7.1%. Heterozygous variants of c.560T.A
stopped after 15 minutes by adding ice-cold methanol. Precipitated proteins and c.736T.A were detected in 2.7% and 0.3% of the samples,

SNPs in mARC
721
TABLE 2
Allele and genotype frequencies of nonsynonymous SNPs in MARC1 and MARC2
SNP (Amino
Acid Change)
Allele
Frequencya
dbSNP Frequencyb
Genotype
Frequencya
dbSNP Frequencyb
%
%
%
%
MARC1
c.493A.G
rs2642438
(T165A)
c.560T.A
rs17850677
(M187K)
c.736T.A
rs3738178
(C246S)
A
G
30.4
69.6
28.3
71.7
A/A
A/G
G/G
T/T
T/A
A/A
T/T
T/A
A/A
G/G
G/C
C/C
7.1
46.6
46.2
97.3
2.7
0.0
99.7
0.3
10.0
36.7
53.3
No data
No data
No data
100.0
0.0
0.0
95.2
4.8
0.0
T
A
98.6
0.4
No data
No data
No data
100.0
T
A
99.8
0.2
0.0
0.0
100.0
0.0
c.739G.C
rs72470601
(D247H)
G
C
100.0
0.0
97.6
2.4
0.0
MARC2
c.730G.A
rs3795535
(G244S)
c.735T.G
rs76664695
(C245W)
G
A
99.5
0.5
99.6
0.4
G/G
G/A
A/A
T/T
T/G
G/G
99.1
0.9
0.0
100.0
0.0
0.0
99.1
0.9
0.0
No data
No data
No data
T
G
100.0
0.0
86.1
13.9
aFrequency data determined in a cohort of 340 healthy Caucasians.
bFrequency data published in dbSNP build 133. Frequency data of rs2642438, rs17850677, rs3738178, rs72470601, and rs3795535
were provided by HapMap.
respectively, whereas no homozygous carriers were identified. c.739G. reduction followed Michaelis–Menten kinetics. Arithmetic means of
C was not detected in the cohort.
For MARC2, only heterozygous variants for c.730G.A were de-
Vmax and Km and their standard deviations are shown in Table 3.
Vmax of mARC-2 variant C245W was significantly decreased
tected in 0.9% of the samples. c.735T.G was not detected in the compared with WT (P , 0.050, ANOVA followed by Dunnett’s post
cohort.
No further variants were found in carriers of the homozygous
hoc test; SigmaPlot 11.0).
Catalytic efficiency was calculated as kcat/Km from kinetic parame-
MARC1 493AA genotype. Two samples of the cohort carried two ters. Arithmetic means of the values and their standard deviations are
heterozygous variants. One sample was heterozygous for c.493A.G shown in Fig. 4.
and c.560T.A in MARC1. Another sample was heterozygous for
c.493A.G in MARC1 and c.730G.A in MARC2.
Catalytic efficiency of mARC-1 WT was 1370 6 430 s21 M21
.
Catalytic efficiency of mARC-2 WT (890 6 150 s21 M21) was
Molybdenum Content of the Recombinant Human mARC statistically significant different compared with mARC-1 WT (P =
Proteins. All protein variants of mARC-1 and mARC-2 were suc- 0.005, t test; SigmaPlot 11.0). Both variants of mARC-2, G244S (580 6
cessfully expressed in E. coli and the WT and variants had similar ex- 40 s21 M21) and C245W (650 6 180 s21 M21), showed a
pression levels.
statistically significant decrease of catalytic efficiency compared
All mARC variants were able to bind Moco. Molybdenum content
of WT and variant mARC protein with one amino acid exchange
could be determined in the range of 14–29 nmol molybdenum per
milligram protein (Fig. 2). The mARC-1 WT and variants showed
a molybdenum content of 22 6 4 nmol molybdenum/mg protein,
representing a molybdenum saturation of 71% 6 14%. The mARC-2
WT and variants had a molybdenum content of 20 6 4 nmol
molybdenum/mg protein, representing a molybdenum saturation of
72% 6 12%. There was no statistically significant difference in
molybdenum content between WT and variant protein (ANOVA fol-
lowed by Dunnett’s post hoc test; SigmaPlot 11.0).
Molybdenum content of mARC-1 WT and variants with more than
one amino acid exchange could be determined in the above-mentioned
range for mARC-1. Values of these protein lots lay between 18 and
26 nmol molybdenum/mg protein (Fig. 3). The mean value of mo-
lybdenum content was 22 6 2 nmol molybdenum/mg protein, repre-
senting a molybdenum saturation of 74% 6 8%.
Kinetic Parameters of mARC WT and Variants. Reduction of
benzamidoxime was carried out with recombinant mARC WT and
variants, cytochrome b5 type B, and NADH cytochrome b5 reductase
and increasing amounts of the substrate in the presence of NADH. The
Fig. 2. Molybdenum content of mARC WT and variants. Molybdenum content was
measured by ICP-MS as described in Materials and Methods. Data are means of
quadruplicates 6 S.D. The black bars present WT proteins, whereas the gray bars
present variant proteins. Mo, molybdenum.

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Ott et al.
Fig. 3. Molybdenum content of mARC-1 WT and multiple variants. Amino acid
substitutions A165T, M187K, V96L, C246S, D247H, and M268I were introduced
successively in mARC-1 WT. Molybdenum content was measured by ICP-MS as
described in Materials and Methods. Data are means of duplicates 6 S.D. The black
bar presents the WT protein, whereas the gray bars present variant proteins.
Fig. 4. Catalytic efficiency of mARC WT and variants. mARC containing 120 pmol
molybdenum, cytochrome b5 type B containing 60 pmol heme, and NADH
cytochrome b5 reductase containing 6 pmol FAD were incubated with 0.1–5 mM
benzamidoxime in 20 mM 4-morpholineethanesulfonic acid, pH 6.0, for 15 minutes
under aerobic conditions. Triplicates of each mARC protein were incubated in
duplicate and measured twice by HPLC. Catalytic efficiency was calculated from
kinetic parameters as described in Materials and Methods. Data are means 6 S.D.
of the number of protein lots mentioned in Table 3. The black bars present WT
proteins, whereas the gray bars present variant proteins.
with mARC-2 WT (P , 0.050, ANOVA followed by Dunnett’s post
hoc test; SigmaPlot 11.0).
N-Reductive Activity of Multiple Variants of mARC-1. Kinetic
parameters were determined for mARC-1 WT and variants, in which
amino acid substitutions A165T, M187K, V96L, C246S, D247H, and
M268I were successively introduced. Vmax decreased almost con-
stantly from WT to the sextuple variant, in which all six amino acids
were exchanged. Km increased almost constantly in this order
(Table 4). Catalytic efficiency also decreased in this order of variants
and was almost absent in the sextuple variant (Fig. 5). Simultaneous
incubation of the WT and all variants confirmed this continuous
decrease in specific N-reductive activity (data not shown).
actually be considered as the WT rather than variant. These data are
consistent with frequency data of several other studies in Caucasian
populations published in the dbSNP. The 735G allele was not detected
in our cohort, although dbSNP build 133 reported an allele frequency
of 13.9%. However, later dbSNP builds removed this information.
Carriers of more than one variant were rare. In carriers of the
homozygous variant MARC1 493AA, no other variants were detected.
Only two subjects carrying two heterozygous variants were observed
in the cohort of 340 healthy Caucasians. One subject was het-
erozygous for c.493A.G and c.560T.A in MARC1. Another was
heterozygous for c.493A.G in MARC1 and c.730G.A in MARC2.
The latest release of the SNP database (dbSNP build 138) published
41 nonsynonymous missense SNPs in MARC1 and 27 in MARC2.
Most of the data were provided from huge sequencing projects.
Frequency data are published for 25 of the 41 nonsynonymous SNPs
in MARC1, but 17 SNPs are absent among Caucasians. Of the
published allele frequencies in MARC2 in Caucasians, frequency data
are published for only three nonsynonymous SNPs in MARC2.
Based on the information on the presence of genetic variants in
MARC1 and MARC2 in Caucasians, we were interested in whether the
proteins had different binding properties of Moco and different
catalytic properties.
All components of the N-reductive enzyme system were expressed
as truncated soluble recombinant proteins in E. coli. The predicted
N-terminal mitochondrial targeting sequences of the mARC proteins,
the membrane-anchoring domain of cytochrome b5 type B, and the
membrane-binding domain of NADH cytochrome b5 reductase were
removed to improve expression (Wahl et al., 2010). Using E. coli
TP1000 as an expression system of mARC proteins ensures that the
eukaryotic form of Moco is expressed (Palmer et al., 1996). The
simple incubation of all three proteins is a useful tool for in vitro
metabolism studies and offers the possibility of analyzing the cat-
alytic ability of recombinant expressed mARC variants until a
Discussion
Genotyping of genetic variants in both mARC genes revealed that
most of them had a low frequency in this Caucasian population. An
exception was MARC1 c.493A.G (rs2642438), which had an allele
frequency of 71.7% for the variant allele; hence, this variant should
TABLE 3
Vmax and Km values of the reduction of benzamidoxime by mARC WT and variants
Data are the means 6 S.D. of three different incubations of each protein lot.
a
a
mARC Protein
Protein Lots
Vmax
Km
n
nmol BA/min per mg protein
mM BAO
mARC-1
mARC-1
V96L
A165T
M187K
C246S
4
1
2
2
2
2
1
105 6 23
138 6 24
125 6 28
104 6 22
93 6 21
0.2 6 0.1
0.3 6 0.1
0.4 6 0.2
0.3 6 0.1
0.2 6 0.1
0.3 6 0.1
0.2 6 0.1
D247H
M268I
113 6 18
94 6 18
mARC-2
mARC-2
G244S
3
1
2
119 6 34
84 6 10
48 6 11b
0.4 6 0.1
0.4 6 0.1
0.2 6 0.1
C245W
BA, benzamidine; BAO, benzamidoxime.
aEach mARC protein was incubated in duplicate and measured twice by HPLC. Kinetic
parameters were determined as described in Materials and Methods.
bStatistical significant decrease of Vmax compared with WT (P , 0.050, ANOVA followed by
Dunnett’s post hoc test; Sigma Plot 11.0)

SNPs in mARC
723
TABLE 4
Moco is the essential basis for N-reductive activity of mARC
proteins (Wahl et al., 2010). For example, 1 mol Moco is bound to
1 mol mARC protein as a prosthetic group (Havemeyer et al., 2011;
Hille et al., 2011). Each Moco contains an oxidized molybdenum atom
in its center and is a highly unstable structure after liberation from the
protein (Schwarz et al., 2009). Molybdenum content of the mARC
proteins can easily be measured by ICP-MS. Results showed that the
mARC WT and variants possess molybdenum, which can only be
derived from Moco because molybdenum was absent in all negative
controls. An mARC WT expressed in the E. coli strain RK5204 and
a NADH cytochrome b5 reductase purified after expression in
a sodium molybdate-containing medium were used as negative
controls. WT and protein variants of mARC-1 and mARC-2 were
successfully expressed in E. coli, but differences in molybdenum
content between the WT and variants were not detectable. Therefore,
we conclude that the investigated nonsynonymous SNPs did not
influence the binding of Moco to the variant protein.
Vmax and Km values of the reduction of benzamidoxime by mARC-1 WT and
multiple variants
Data are the means 6 S.D.
b
b
mARC Proteina
Protein Lots
Vmax
Km
n
nmol BA/min per mg protein
mM BAO
mARC-1
2
1
1
1
1
1
1
106 6 25
82 6 1
110 6 1
62 6 2
52 6 1
41 6 5
1 6 0
0.2 6 0.0
0.2 6 0.0
0.3 6 0.0
0.5 6 0.0
0.4 6 0.0
0.4 6 0.2
0.7 6 0.2
Single variant
Double variant
Triple variant
Quadruple variant
Quintuple variant
Sextuple variant
BA, benzamidine; BAO, benzamidoxime.
aAmino acid substitutions A165T, M187K, V96L, C246S, D247H, and M268I were
introduced successively in mARC-1 WT.
bEach mARC protein was incubated in duplicate and measured twice by HPLC. Kinetic
parameters were determined as described in Materials and Methods.
Kinetic parameters of all mARC variants were determined. Steady
state assays revealed that mARC-1 and mARC-2 follow Michaelis–
Menten kinetics.
Statistically significant decreases of kinetic parameters were only
detected in variants of mARC-2. The Vmax of the C245W variant and
catalytic efficiency values of the G244S and C245W variant (P ,
0.050, ANOVA followed by Dunnett’s post hoc test; SigmaPlot 11.0)
were lower than in the WT. However, subjects carrying any
homozygous variant genotype leading to the protein variants G244S
and C245W were not detected in our sample.
Km values of human mARC-2 were slightly higher than that of
human mARC-1. This led to a statistically significant increase of
catalytic efficiency of mARC-1 WT compared with mARC-2 WT (P =
0.005, t test; SigmaPlot11.0). Human mARC-1 seems to be the
isoform of higher affinity and catalytic efficiency toward benzami-
doxime. This is consistent with results of previous studies (Wahl et al.,
2010; Krompholz et al., 2012). Catalytic efficiency is not influenced
by variants of mARC-1. There were no statistically significant dif-
ferences in kinetic parameters between mARC-1 WT and mARC-1
variants. In addition, the A165T variant of mARC-1, which was
detected in our sample, did not show any different kinetic parameters.
Because a single SNP in MARC1 was not able to induce a loss of
function in the mARC-1 protein, multiple variants were recombinantly
expressed to investigate whether they exert greater influence on the
N-reductive activity. Indeed, analysis of multiple variants of mARC-1
showed an almost constant decrease in the catalytic efficiency from
the WT to sextuple variants (Fig. 5).
three-dimensional structure is available that shows how the enzyme
system is anchored in the outer mitochondrial membrane (Havemeyer
et al., 2011).
Similar in vitro metabolism studies were performed successfully
with another molybdenum enzyme, human sulfite oxidase. Expression
of recombinant human sulfite oxidase, in which its mitochondrial
targeting signal was also removed, was optimized in E. coli TP 1000,
leading to a high amount of active protein. Proteins with or without the
His-tag did not show any differences in kinetic parameters (Temple
et al., 2000). The R160Q variant causes sulfite oxidase deficiency in
vivo. Expression of this variant by site-directed mutagenesis revealed
a protein that was able to bind the prosthetic groups, Moco and heme,
but showed a 1000-fold decrease of catalytic efficiency with the
substrate sulfite in vitro (Garrett et al., 1998).
After replacements of more than two amino acids in mARC-1,
catalytic efficiencies were at least 5-fold decreased compared with the
WT. This result suggests that more than two amino acids have to
be changed in the variant mARC protein to induce differences in
N-reductive activity.
In sextuple variants, N-reductive activity was almost absent al-
though Moco was bound to the protein. Possibly all of the inves-
tigated amino acid changes did not influence the binding of Moco but
together might participate in binding of the substrate benzamidoxime.
The double variant of mARC-2, which contained the amino acid
Fig. 5. Catalytic efficiency of mARC-1 WT and multiple variants. Amino acid
substitutions A165T, M187K, V96L, C246S, D247H, and M268I were introduced changes G244S and C245W, was expressed in E. coli and was found
successively in mARC-1 WT. mARC containing 120 pmol molybdenum,
cytochrome b5 type B containing 60 pmol heme, and NADH cytochrome b5
reductase containing 6 pmol FAD were incubated with 0.1–5 mM benzamidoxime in
to bind Moco. Catalytic efficiency was not further decreased as
observed for the C245W variant (data not shown).
The mARC enzyme system is important for drug development and
detoxification. On the one hand, the N-reductive enzyme system is
able to activate prodrugs with an amidoxime structure (Clement,
2002). Oral bioavailability is important for the drug compliance of
patients and helps reduce costs in health care systems compared with
20 mM 4-morpholineethanesulfonic acid, pH 6.0, for 15 minutes under aerobic
conditions. Each mARC protein was incubated in duplicate and measured twice by
HPLC. Catalytic efficiency was calculated from kinetic parameters as described in
Materials and Methods. Data are means 6 S.D. of two lots of WT and means of one
lot of each variant. The black bar presents the WT protein, whereas the gray bars
present variant proteins.

724
Ott et al.
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any interindividual variability in activation are needed. On the other
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Authorship Contributions
Participated in research design: Ott, Reichmann, Boerger, Cascorbi, Bittner,
Mendel, Kunze, Clement, Havemeyer.
Contributed new reagents or analytic tools: Cascorbi.
Performed data analysis: Ott, Boerger.
Wrote or contributed to the writing of the manuscript: Ott, Cascorbi, Bittner,
Clement, Havemeyer.

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