NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its biological role

Article abstract of DOI:10.1016/j.febslet.2010.08.019

NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide and an NAD⁺ degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to rapidly scavenge H₂O₂ produced during the oxidative process or added exogenously. Structured summary: MINT- 7990140: nfrA1 (uniprotkb:. P39605) and nfrA1 (uniprotkb:. P39605) bind (MI:. 0407) by X-ray crystallography (MI:. 0114).

Full text of DOI:10.1016/j.febslet.2010.08.019

FEBS Letters 584 (2010) 3916–3922
journal homepage: www.FEBSLetters.org
NADH oxidase activity of Bacillus subtilis nitroreductase NfrA1: Insight into its
biological role
a
b
a
c
d
Sylvie Cortial , Philippe Chaignon , Bogdan I. Iorga , Stéphane Aymerich , Gilles Truan ,
e
e
e
a,
⇑
Virginie Gueguen-Chaignon , Philippe Meyer , Solange Moréra , Jamal Ouazzani
a
Institut de Chimie des Substances Naturelles (ICSN), C.N.R.S. UPR 2301, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
Laboratoire de chimie et biochimie des microorganismes, Université de Strasbour, 1 rue Blaise Pascal F-67070 Strasbourg, France
Microbiologie et Génétique Moléculaire, INRA (UMR1238) CNRS (UMR2585), AgroPariTech, F-78850 Thiverval-Grignon, France
Centre de Génétique Moléculaire (CGM), Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France
b
c
d
e
Laboratoire d’Enzymologie et Biochimie Structurales (LEBS), CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
NfrA1 nitroreductase from the Gram-positive bacterium Bacillus subtilis is a member of the
NAD(P)H/FMN oxidoreductase family. Here, we investigated the reactivity, the structure and kinetics
of NfrA1, which could provide insight into the unclear biological role of this enzyme. We could show
that NfrA1 possesses an NADH oxidase activity that leads to high concentrations of oxygen peroxide
Received 23 July 2010
Revised 6 August 2010
Accepted 12 August 2010
Available online 18 August 2010
+
and an NAD degrading activity leading to free nicotinamide. Finally, we showed that NfrA1 is able to
2 2
rapidly scavenge H O produced during the oxidative process or added exogenously.
Edited by Miguel De la Rosa
Structured summary:
Keywords:
MINT-7990140: nfrA1 (uniprotkb:P39605) and nfrA1 (uniprotkb:P39605) bind (MI:0407) by X-ray crystal-
lography (MI:0114)
Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Nitroreductase
NADH oxidase
Hydrogen peroxide
NAD+ degradation
Crystal structure
Bacillus subtilis
1
. Introduction
Nitroreductases are members of the NAD(P)H/FMN oxidoreduc-
further oxidation and ring scission [6]. Nitroreductases catalyze
the reduction of Cr(VI) to the less toxic Cr(III) compounds [7] and
the reduction of various azo dyes in the presence of additional
external FMN [8]. More recently, nitroreductases were shown to
be involved in high-value projects such as prodrug activation in
gene-directed anticancer therapies [9], the activation of hypoxic
anticancer nitro drugs in solid tumors [10], and enzyme-based bio-
sensors for nitro-sensitive compounds [11,12].
tase family. This class of enzymes has the remarkable ability to cat-
alyze one- or two-electron transfers through ionic or radical
mechanisms [1,2], and catalyze hydride or oxygen transfer to or
from various organic compounds [3].
Nitroreductases were first identified as targets for the eradica-
tion of various pathogens by nitrofuran and nitroimidazole antibi-
While NAD(P)H/FMN oxidoreductases are ubiquitous and
essential enzymes, the biological roles of nitroreductases have
not yet been elucidated. Recent investigations classified the nitro-
reductase from Bacillus subtilis [13] among the heat shock proteins.
Ò
otics. Among them, nifurtimox (Lampit ), nitrofurantoin
Ò
Ò
(
[
Furadantin ), and metronidazole (Flagyl ) are still widely used
4]. The pathogen’s nitroreductases catalyze the reduction of the
+
nitro group to the corresponding genotoxic hydroxylamine. Resis-
tant strains have been shown to downregulate or lack the expres-
sion of this enzyme [5]. Nitroreductases are also involved in the
bioremediation of nitroaromatic and nitroheterocyclic explosives.
These reactions lead to primary amine derivatives that undergo
NADH oxidases catalyze the oxidation of NADH to NAD using
molecular oxygen as the electron acceptor. They have recently
gained interest for their involvement in many biological defects,
including aging-related diseases [14], atherosclerosis [15], apopto-
sis [16], endothelium-dependent vasodilation and retina damage
in diabetes [17], and antibiotic, antiparasitic and antifungal resis-
tance. NADH oxidases have also been used in biocatalysis for
+
NAD recycling coupled to secondary alcohol or amino acid oxi-
dases. Some NADH oxidases are also peroxiredoxin oxidoreduc-
tases [18] that are involved not only in the regeneration of NAD
but also in the removal of peroxides.
⇑
Corresponding author. Fax: +33 169077247.
E-mail address: Jamal.ouazzani@icsn.cnrs-gif.fr (J. Ouazzani).
0
014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2010.08.019

S. Cortial et al. / FEBS Letters 584 (2010) 3916–3922
3917
In this paper, we investigated the structure and kinetics of the
2.6. Superoxide measurement
Bacillus nitroreductase NfrA1. The protein structure determined
by X-ray crystallography at 1.95 Å resolution resembles the topol-
ogy of the NADH oxidase superfamily. With the aim of better
understanding the biological role of this enzyme, we showed for
the first time that NfrA1 exhibits a strong NADH oxidase activity,
Superoxide was measured according to published methods [20],
and results compared to a xanthine/xanthine oxidase control
(600 lM xanthine + 2.6 ll xanthine oxidase 0.1 U).
+
catalyzes the degradation of NAD and scavenges high concentra-
2.7. Nicotinamide quantification
2 2
tions of H O . Our data offer new perspectives for the further
development of this class of enzyme.
Nicotinamide was quantified by HPLC. The chromatography
system consisted of an Alliance 2695 module and a 2996 photodi-
ode array detector (PDA), both controlled by Millennium software
2
. Materials and methods
(Waters corporation). The Hypercarb column (5
l
m, 100 Â 4.6 mm
id; ThermoQuest) was eluted at 1 ml/min for 50 min using a linear
gradient from water (0.1% formic acid) to acetonitrile (0.1% formic
acid).
2.1. Strains
Bacillus LMA was recovered from industrial aqueous waste (CEA,
Le Ripault, Monts, France). The strain was deposited in the CNCM
collection (Institut Pasteur, France) and referenced as Bacillus
CNCM I-1915.
2.8. Crystallographic analysis
Commercial crystallization solutions (Qiagen kits) were
screened using a nanodrop Cartesian robot (Proteomic Solutions)
at 293 K. The condition 90 from the Classics suite was manually
optimized in hanging drops composed of 1:1 volume ratio of crys-
tallization condition and seleniated NfrA1 at 5 mg/ml. Crystals ob-
tained in 0.1 M Tris–HCl pH 7.6, 30% (w/v) PEG 4000, 200 mM
sodium acetate were flash-frozen in a cryo-protecting solution
consisting of the mother solution supplemented with 20% (v/v)
glycerol.
2.2. Reagents
All chemicals were of the highest commercially available grade
and purchased from Sigma–Aldrich, Fluka, or Merck (Lyon, France).
2.3. Cloning, expression, and purification of B. subtilis nfrA1
Based on nfrA1 gene of the standard Bacillus 168 strain, the nfrA1
A single-wavelength anomalous diffraction experiment was
carried out at 100 K on ID14-H4 at the European Synchrotron Radi-
ation Facility (ESRF, Grenoble, France). Diffraction intensities were
integrated using XDS [21].
gene of Bacillus LMA was amplified by polymerase chain reaction
PCR) with genomic DNA as template. The following primers were
used:
(
The seleniated NfrA1 structure was determined by the SAD
method. Solvent content analysis using the CCP4 program suite
[22] indicated the presence of four protein monomers forming
two dimers in the asymmetric unit, which corresponded to a Mat-
thews coefficient [23] of 2.12 Å Da and a solvent content of
42.1%. The positions of 24 selenium atoms were found in the
asymmetric unit (6 atoms/monomer) using SHELX [24]. The phases
were calculated using SHELXE [24] and RESOLVE [25] was used
to perform solvent-flatening and non-crystallographic symmetry
nfrA1-Bam: CGG GGA TCC ATG AAT AAC ACA ATC GAA ACC;
nfrA1-Hind: CCG AAG CTT GTT TTT ATT AAA GCC CTT TTC;
3
À1
(
restriction sites are underlined). The pQE-30 vector allows the
expression of the protein with a hexahistidine affinity tag at the
N-terminus to facilitate purification. The construct was trans-
formed into the expression host Escherichia coli M15-pREP4 (Qia-
gen). Cells were grown at 37 °C to an OD600 of 0.6, and 1 mM
isopropyl-b-D-thiogalactopyranoside was added to induce the pro-
duction of His-tagged NfrA1 over a period of 3 h. After centrifuga-
tion, pellets were washed in 40 mM Tris–HCl, pH 7.4 and lysed on
a Dyno-Mill homogenizer (GB Mill). Ultracentrifugation superna-
tants were purified through a His-Trap HP affinity column (GE-
healthcare). The pooled nitroreductase fractions were loaded onto
the column and eluted using 80 column volumes of a linear imidaz-
ole gradient (0–500 mM) in 25 mM Tris–HCl, pH 7.4 and 0.5 M
NaCl. This standard procedure yielded 10 mg of NfrA1 per gram of
wet biomass.
Table 1
Crystallographic data and refinement parameters.
PDB code
3N2S
Space group P1
Cell parameters (Å,°)
Wavelength (Å)
Resolution (Å)
No. of observed reflections
a = 52.5 b = 54.8 c = 90.5
a = 87.7 p = 87.6 y = 65.1
0.9792
20–1.95 (2–1.95 Å)
499 279
127 396
3.7 (11.9)
95.5 (86.6)
23.8 (9.9)
17.3
2.4. SDS–PAGE and protein determination
No. of unique reflections
a
Rsym (%)
Completeness (%)
These analysis were carried out according to published methods
I/
r
[
19]. Gels were stained with Coomassie Brilliant Blue R.
cryst, (%)b
R
R
c
free (%)
19.8
2
.5. Enzyme essays
rms bond deviation (Å)
0.01
1.03
25
13.3
rms angle deviation (°)
2
Average B (Å )
The enzyme essay consisted of hydrogen peroxide measure-
Protein
FMN cofactor solvent
ment. Briefly, 67
5
lM NfrA1 was mixed with 2 mM NADH in
26.6
0 mM ammonium bicarbonate, pH 8.4 and placed at 37 °C. Par-
a
R
sym
=
R
hkl
R
i
|
I
i
(hkl) À <I(hkl)>|/
i i i
Rhkl R I (hkl), where I (hkl) is the ith
tial anaerobia was established by air replacement with argon, vac-
uum–purge cycles and application of nitrogen flux. Hydrogen
peroxide production was determined colorimetrically using the
PeroXOquant Quantitative Peroxide Assay Kit (Pierce, Rockford,
IL).
observed amplitude of reflection hkl and <I(hkl)> is the mean amplitude for all
observations i of reflection hkl.
b
R
cryst
=
R
||Fobs| À |Fcalc||
Five percent of the data were set aside for free R-factor calculation values for the
highest resolution shell are in parentheses.
R|Fobs|.
c

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S. Cortial et al. / FEBS Letters 584 (2010) 3916–3922
3
. Results and discussion
.1. Structure of NfrA1
The structure of NfrA1 was determined using the SAD method
3
and seleniated protein. The asymmetric unit of the NfrA1 crystal
contains two similar homodimers (dimers AB and CD) of interlock-
ing subunits with an average root mean square deviation (rmsd) of
0
.55 Å for all Ca atoms (Fig. 1). A structural comparison of the
NfrA1 dimer to all entries in the PDB indicated that the three most
similar overall structures are the NADPH:FMN reductase of Vibrio
harveyi (PDB 1BJK and 2BJK) [33,34], E. coli oxygen-insensitive
nitroreductase NfsA [35] (PDB 1F5V) and YcnD from B. subtilis
[
(
36] (PDB 1ZCH), NADH oxidase from Thermus thermophilus [37]
PDB 1NOX) and oxidoreductase of V. fischeri [38] (FRaseI; PDB
1
VRF). Eight of the 10 NfrA1 residues involved in FMN binding
are conserved between Frp, NfsA, NfrA2 and NfrA1 (Fig. 2) [39].
3.2. NADH oxidase activity of NfrA1
Nitroreductases can be distinguished according to their inhibi-
tion by oxygen [35]. To evaluate the sensitivity of NfrA1 to oxygen,
we compared the reduction of various nitro substrates listed in
(Fig. 3) under aerobic and partially anaerobic conditions. The HPLC
profiles showed no significant differences, suggesting that oxygen
failed to inhibit NfrA1 (data not shown). Insensitivity to oxygen
could be due to a lack of reactivity with oxygen or attributed to
its fast reduction. Therefore, in the absence of nitro substrates,
hydrogen peroxide and superoxide ions were monitored using
classical colorimetric and fluorometric techniques. A rapid produc-
tion of more than 200
served, followed by a slow decrease that stabilized around
25 M within 60 min (Fig. 4A). H production is drastically
inhibited under partial anaerobia. In this reaction no superoxide
ion was formed (Fig. 4B). These observations were supported by
the removal of hydrogen peroxide by catalase, while superoxide
dismutase had no effect (Fig. 4C). In conclusion, NfrA1 is a potent
NADH oxidase that is able to produce a high concentration of
2 2
lM H O during the first 5 min was ob-
1
l
2 2
O
Fig. 1. Ribbon representation of the two NfrA1 dimers in the asymmetric unit (in
dimer AB and molecule D, the -helices are colored cyan and the b-sheets are
colored magenta, while in molecule C they are in red and yellow, respectively). Loop
11–218 (green) of the excursion domain of molecule C enters the substrate-
a
2
binding site of molecule A. FMN cofactors are shown in orange stick form.
2 2
H O useful for the defense of Bacillus strains against microbial
competition [40]. Bacillus strains are highly resistant to hydrogen
peroxide due to efficient catalase activity [41]. Furthermore, as
averaging and build a first very limited protein model. Arp-warp
[
26] permitted the building of approximately 80% of each chain.
At this stage, the electron density map was of high enough quality
for the two dimers to be built manually using TURBO-FRODO [27].
The atomic model was refined using CNS [28] and BUSTER-TNT
shown in Fig. 5, NfrA1 exerts a dual role by producing H
the invading strains and scavenging H into the producing strain.
Fig. 5 shows the rapid and efficient decrease in exogenous H . In
the absence of NADH; H concentration decreased from 200 to
M in 10 min and was completely removed in about 1 h. In
the presence of NADH and 200 M exogenous H , NfrA1 exerted
its NADH oxidase activity by converting oxygen into additional
2 2
O against
2 2
O
2 2
O
[
29]. Data collection and refinement statistics are given in
2 2
O
Table 1.
60 l
l
2 2
O
2.9. Docking experiments
H
1
2
O
2
(350
lM), followed by a slow decrease to 285 lM within
All docking studies were performed using Glide docking soft-
h. In the absence of NfrA1, hydrogen peroxide concentration
ware (version 5.5) [30,31] and the standard parameters within
the SP procedure without any constraints. The figure showing the
docking complex was generated using Chimera [32].
was not affected. In the absence of NADH, the activity of NfrA1
could be explained by the direct conversion of the high accessible
methionine residues to the corresponding sulfoxides. NfrA1 count
Fig. 2. Structure-based sequence alignment of NfrA1 (PDB code 3N2S) with Frp (1BKJ), NfsA (1F5 V) and NfrA2 (1ZCH) using ESPript [39]. Positions of the NfrA1 residues
involved in FMN and NAD binding are indicated by red triangles and green stars, respectively.

S. Cortial et al. / FEBS Letters 584 (2010) 3916–3922
3919
Fig. 3. NfrA1 catalyzes the reduction of diverse nitro substrates to their corresponding primary amines.
Fig. 4. (A) Production of hydrogen peroxide by NfrA1 + NADH under aerobic conditions (black circles), partial anaerobic conditions (black triangles) and without NADH
white circles). (B) Production of superoxide ion with NfrA + NADH (gray line) and with the xanthine–xanthine oxidase control. (C) The production of hydrogen peroxide by
NfrA + NADH was confirmed by catalase (13 U/ml) or superoxide dismutase (10 U/ml) additions followed by hydrogen peroxide measurement at 5 min (black column) and
5 min (white column).
(
1
six Met residues per monomer, four are easily accessible, Met-128,
98 and 229 on the surface of the protein and Met-106 in the cat-
alytic site. Such an interaction has been widely reported in the lit-
erature [42].
1

3
920
S. Cortial et al. / FEBS Letters 584 (2010) 3916–3922
syltransferases) [47]. According to our results, hydrogen peroxide
is the only reactive oxygen species formed by NfrA1. Hydroxyl rad-
ical, the most reactive agent, could not form, as no superoxide ion
was detected (Fig. 6) [2,49].
+
Therefore, 200 and 400
l
M H
2
O
2
were incubated with NAD un-
der the same conditions as the enzymatic reaction. However, no
nicotinamide was formed (Fig. 7). This result suggests that NAD+
is probably cleaved in the catalytic site of the enzyme by a highly
oxidizing reagent that could be peroxyflavin (Fig. 8). No evidence
for such a reaction has been previously presented in the literature,
+
suggesting a potentially new mechanism of NAD degradation. To
check the validity of this hypothesis, molecular docking was used
to investigate whether the positioning of peroxy-FMN and NAD+
in the binding site of the NfrA1 structure is compatible with the
reaction observed experimentally. Even though most of the dock-
ing software currently available is unable to simultaneously dock
Fig. 5. NfrA scavenging of exogenous hydrogen peroxide (triangles). This reaction
was NADH-independent and was also observed with cumulating exogenous and
endogenous hydrogen peroxide (Lozenge).
+
3
.3. Degradation of NAD by NfrA1
During the screening of nitro substrates, the formation of a
common peak at retention time 9 min with a molecular weight
of 122 D was noted. The production of this compound was drasti-
cally inhibited under partial anaerobia. This compound was iso-
+
lated and identified as nicotinamide, suggesting that the NAD
cofactor is cleaved during the reaction. Molecules and enzymes
that catalyze the degradation of nicotinamide cofactors are in-
volved in major biological disorders [43]. They include toxins
(
diphtheria and cholera toxins) [44] and the Sir-2 enzymes in-
volved in gene silencing, DNA repair, and cell longevity [45].
+
NAD degradation is also catalyzed by immunological factors
(
[
CD38 and CD157) [46] and involved in ADP-ribosylation reactions
47]. Two types of catalysts are known to induce such a degrada-
Fig. 7. Free nicotinamide is formed during the enzymatic reaction (NR + NADH) but
not in the presence of exogenous hydrogen peroxide. Concentrations were deduced
from the HPLC peak surface reported on a standard linear plot (right plot).
tion: reactive oxygen species [48] and hydrolytic enzymes (ribo-
Fig. 6. Reduced flavin interacts with molecular oxygen, leading to a variety of highly reactive oxidizing agents such as peroxyflavin.

S. Cortial et al. / FEBS Letters 584 (2010) 3916–3922
3921
Fig. 8. Proposed mechanism for the oxidation of NAD+ by the peroxyflavin form of FMN in NfrA1 and production of free nicotinamide.
the Gif campus (http://www.imagif.cnrs.fr). B.I. Iorga thanks David
Rinaldo and Katia Dekimeche (Schrödinger LLC) for help with set-
ting up the docking protocol.
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Fig. 9. Docking conformations of peroxy-FMN and NAD+ in the NfrA1 binding site.
[
[
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+
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In conclusion NfrA1 has probably a dual role in the regulation of
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activity, NfrA1 produce high concentrations of H O that can serve
2 2
as a defense agent against invading strains. In contrast, and due to
abundant and accessible methionine residues NfrA1 can also scav-
2 2
enge H O , probably by converting these residues to the corre-
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degradation of NAD+ to free nicotinamide trough an oxidative
catalysis involving probably the peroxyflavin intermediate. Such
a mechanism of nicotinimide cofactor regulation have never been
reported before.
[
[
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Data bank accession code
The atomic coordinates and structure factors of NfrA1 have
been deposited in the protein data bank (http://www.rcsb.org) un-
der accession code 3N2S.
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We thank the staff of ESRF in Grenoble for making station ID14-
H4 available. The crystallization work benefited from the LEBS
facilities of the IMAGIF Structural Biology and Proteomic Unit at
[
18] Poole, L.B., Higuchi, M., Shimada, M., Calzi, M.L. and Kamio, Y. (2000)
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