Báo cáo khoa học: Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis potx

Characterization of L-aspartate oxidase and quinolinate
synthase from Bacillus subtilis
Ilaria Marinoni
1
, Simona Nonnis
2
, Carmine Monteferrante
1
, Peter Heathcote
3
, Elisabeth Ha
¨
rtig
4
,
Lars H. Bo
¨
ttger
5
, Alfred X. Trautwein
5
, Armando Negri
2
, Alessandra M. Albertini
1
and
Gabriella Tedeschi
2
1 Department of Genetics and Microbiology, University of Pavia, Italy
2 D.I.P.A.V., Section of Biochemistry, University of Milano, Italy
3 School of Biological and Chemical Sciences, Queen Mary College, University of London, UK

4 Institute of Microbiology, Technical University of Braunschweig, Germany
5 Institute of Physics, University of Lu
¨
beck, Germany
NAD is a ubiquitous and essential molecule in all
living organisms. In addition to its well-established
role in redox biochemistry and energetic metabolism,
NAD can function as a signaling molecule in a variety
of cellular processes [1]. In eubacteria, NAD is pro-
duced by a de novo pathway or starting from pre-
formed nicotinic acid. Quinolinic acid is the precursor
for the de novo pathway; in most eukaryotes, it is pro-
duced via degradation of tryptophan, whereas in many
eubacteria, including several pathogens, it is synthe-
sized from l-aspartate and dihydroxyacetone phos-
phate (DHAP). This reaction involves the so-called
quinolinate synthase complex: the first enzyme,
l-aspartate oxidase (NadB, EC 1.4.3.16), encoded by
the gene nadB, catalyzes the oxidation of l-aspartate
to iminoaspartate; the second enzyme, quinolinate syn-
thase (NadA), is encoded by the gene nadA and cata-
lyzes the condensation between iminoaspartate and
DHAP, resulting in quinolinic acid production
(Scheme 1) [2]. Quinolinic acid is then converted to
Keywords
L-aspartate oxidase; NAD biosynthesis;
NadA; NadB; quinolinate synthase
Correspondence
G. Tedeschi, D.I.P.A.V., Section of
Biochemistry, University of Milano, Via

Celoria 10, 20133 Milano, Italy.
Fax: +39 02 50318123
Tel: +39 02 50318127
E-mail:
(Received 4 July 2008, revised 1 August
2008, accepted 12 August 2008)
doi:10.1111/j.1742-4658.2008.06641.x
NAD is an important cofactor and essential molecule in all living organ-
isms. In many eubacteria, including several pathogens, the first two steps in
the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB)
and quinolinate synthase (NadA). Despite the important role played by
these two enzymes in NAD metabolism, many of their biochemical and
structural properties are still largely unknown. In the present study, we
cloned, overexpressed and characterized NadA and NadB from Bacil-
lus subtilis, one of the best studied bacteria and a model organism for low-
GC Gram-positive bacteria. Our data demonstrated that NadA from
B. subtilis possesses a [4Fe–4S]
2+
cluster, and we also identified the cysteine
residues involved in the cluster binding. The [4Fe–4S]
2+
cluster is coordi-
nated by three cysteine residues (Cys110, Cys230, and Cys320) that are
conserved in all the NadA sequences reported so far, suggesting a new non-
canonical binding motif that, on the basis of sequence alignment studies,
may be common to other quinolinate synthases from different organisms.
Moreover, for the first time, it was shown that the interaction between
NadA and NadB is not species-specific between B. subtilis and Escherichia
coli.
Abbreviations

DHAP, dihydroxyacetone phosphate; GST, glutathione S-transferase; GST–NadA, quinolinate synthase fused to glutathione S-transferase
(GST) at its N-terminus; IPTG, isopropyl thio-b-
D-galactoside; NadA, quinolinate synthase; NadA–His, quinolinate synthase with a His
6
-tag at
the N-terminus; NadB,
L-aspartate oxidase.
5090 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
nicotinic acid and, finally, to NAD by a biosynthetic
pathway common to all organisms. As NadA and
NadB are absent in mammals, they are considered to
be ideal targets for the development of novel prophy-
lactic and therapeutic agents [3]. Moreover, very
recently, Pruinier et al. [4] reported that the pathogenic
bacterium Shigella is a nicotinic acid auxotroph,
unable to synthesize NAD via the de novo pathway,
due to nadA and nadB gene mutations. When the func-
tionality of nadA ⁄ B in Shigella was restored, a consis-
tent loss of virulence and inability to invade host cells
were observed. On the basis of this result, they defined
NadA and NadB as antivirulence loci.
Besides being important in bacteria, NadA and
NadB analogs seem to be involved in NAD biosynthe-
sis also in plants. Many experimental findings, together
with the apparent absence of genes encoding enzymes
involved in other possible routes to quinolinate, sug-
gest that several plants may obtain this key precursor
via the aspartate pathway, like many bacteria [5–7].
Therefore, because of the growing amount of evi-
dence indicating the importance in several organisms

of de novo NAD biosynthesis through the reaction
catalyzed by NadA and NadB, it is of the utmost
importance to gain a thorough knowledge of the bio-
chemical and structural properties of these two
enzymes.
The gene nadB is present in several microorganisms
and in plants, but the protein has been purified only
from Escherichia coli, Pyrococcus horikoshii and Sulfol-
obus tokadaii, and characterized from a biochemical
and structural point of view only from E. coli and
S. tokadaii [8–17]. It is a flavoprotein containing 1 mol
of noncovalently bound FAD ⁄ mol of protein. This
enzyme presents several peculiarities that distinguish it
from all other flavo-oxidases: (a) in vitro, it is able to
use different electron acceptors such as oxygen, fuma-
rate, cytochrome c and quinones [9], suggesting that it
is involved in NAD biosynthesis in anaerobic as well
as aerobic conditions; and (b) the primary and tertiary
structures are not similar to those of other flavo-oxid-
ases, but to those of the flavoprotein subunit of the
succinate dehydrogenase ⁄ fumarate reductase class of
enzymes. As a consequence, NadB shares with these
proteins most of the active site features, including the
presence of an arginine playing an acid–base role in
catalysis [11–15]. Accordingly, NadB can reduce fuma-
rate, but it is unique in that it is able to stereospecifi-
cally oxidize l-aspartate and is unable to oxidize
succinate.
Interestingly, in 2003, Yang et al. [18] described
another enzyme, from Thermotoga maritima, that is

involved in the de novo biosynthesis of NAD and that
plays the same role as NadB, although it does not
share any recognizable sequence similarity to NadB. It
is described as NADP-dependent l-aspartate dehydro-
genase, and is strictly specific for l-aspartate. This
enzyme produces iminoaspartate, which is then con-
verted to quinolinate through the condensation with
DHAP catalyzed by NadA.
The second enzyme involved in the de novo biosyn-
thesis of NAD, NadA, is extremely sensitive to oxygen;
therefore, it has been poorly characterized so far, and
very little is known regarding its biochemical and
structural properties. The enzyme has only been puri-
fied from E. coli [19–21] and P. horikoshii [22]. Recent
studies on the enzyme from E. coli have demonstrated
that the protein harbors a [4Fe–4S]
2+
cluster [20,21]
that, as it is very sensitive to oxygen, probably explains
why NadA is identified as the site of oxygen poisoning
of NAD synthesis in anaerobic bacteria [23]. The 3D
structure has been obtained for the enzyme from
P. horikoshii [22]. The protein shows a triangular
architecture in which conserved amino acids determine
three structurally homologous domains. Unfortunately,
the structure lacks any data on the [Fe–S] center, and
the three surface loops that contain two highly
conserved cysteine residues are disordered. Moreover,
Scheme 1. Reaction catalyzed by the ‘quinolinate synthase complex’. The first enzyme, NadB, catalyzes the oxidation of L-aspartate to
iminoaspartate using either oxygen or fumarate as electron acceptor for FAD reoxidation; the second enzyme, NadA, catalyzes the conden-

sation between iminoaspartate and DHAP, resulting in quinolinic acid production.
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5091
the canonical binding motif for [4Fe–4S]
2+
clusters
(CXXCXXC) that is found in the C-terminal regions
of most quinolinate synthases from bacteria, including
E. coli [19–21], is absent in NadA from P. horikoshii,
and in this case the cofactors remain to be identified
[22]. The absence of the consensus sequence for the
binding of a [4Fe–4S]
2+
cluster is observed also in
NadA from several plants. On the other hand, very
recently Murthy et al. [7] described a new SufE-like
protein from Arabidopsis thaliana chloroplasts that
contains two domains, one SufE-like domain and one
with similarity to the bacterial NadA carrying a highly
oxygen-sensitive [4Fe–4S]
2+
cluster. Therefore, two
important areas have to be clarified: (a) the nature of
the cofactor for quinolinate synthase, in particular for
NadA proteins that do not contain a canonical binding
motif for a [4Fe–4S]
2+
cluster; and (b) the identifica-
tion of the residues involved in the binding of the
[4Fe–4S]

2+
cluster, if present.
In an attempt to resolve some of these issues, we
cloned, overexpressed and characterized NadA and
NadB from Bacillus subtilis, one of the best studied
bacteria and a model for low-GC Gram-positive bacte-
ria, including pathogens. Our data add new informa-
tion regarding the NadA cofactor and the interaction
between NadA and NadB. In particular, it is demon-
strated that the cofactor for NadA from B. subtilis is a
[4Fe–4S]
2+
cluster, even though the sequence does not
show a canonical binding motif. Moreover, for the first
time, the cysteines involved in the cluster binding are
identified. Taken together, our data suggest that in
NadA from B. subtilis, the [4Fe–4S]
2+
cluster is coor-
dinated by three strictly conserved cysteine residues
(Cys110, Cys230, and Cys320). Thus, NadA presents a
new noncanonical binding motif that, on the basis of
sequence alignment studies, may be common to other
quinolinate synthases from different sources. More-
over, the results show for the first time that the inter-
action between NadA and NadB is not species-specific
between the proteins from B. subtilis and E. coli.
Results and Discussion
NadA cloning and protein purification and
characterization

In order to optimize the heterologous production and
purification of B. subtilis NadA, several expression
vectors with different tags were utilized: NadA with a
His
6
-tag at the N-terminus, NadA with a His
6
-tag at
the C-terminus (NadA–His), and NadA fused to gluta-
thione S-transferase (GST) at its N-terminus (GST–
NadA). The best results in terms of soluble protein
yield were obtained by cloning the nadA gene in
pET28-a with the His-tag at the C-terminal region.
Upon purification in a glove box under anaerobic con-
ditions, a soluble pure protein, brown in color, was
obtained with a yield of 10 mg of pure protein from
1L of E. coli culture expressing B. subtilis NadA
(Fig. 1A). Therefore, this protein was utilized for
further studies. As determined by gel filtration, it is a
trimer of 124 kDa (expected molecular mass for the
monomer 41 kDa) under both aerobic and anaerobic
conditions (data not shown).
To evaluate its enzymatic activity, quinolinate for-
mation was measured by a discontinuous enzymatic
assay that couples the production of iminoaspartate by
NadB with the condensation between DHAP and
iminoaspartate to form quinolinic acid catalyzed by
NadA [19] (Scheme 1). As described below, NadB is
able to use both molecular oxygen and fumarate as
electron acceptors for FAD reoxidation. Therefore, to

better evaluate NadA activity, the assays were per-
formed under aerobic and anaerobic conditions (in the
presence of fumarate), using recombinant B. subtilis
NadA plus B. subtilis NadB, overexpressed and puri-
fied as detailed below. Different concentrations of
NadA, NadB and fumarate (under anaerobic condi-
tions) were utilized in order to set up a suitable assay
to be used to check NadA activity. The data showed
that: (a) the assay is linear up to 0.25 mg of NadA;
(b) 10 lg of NadB is the lowest amount suitable to
measure NadA activity; and (c) under anaerobic condi-
tions, NadA activity becomes independent of fumarate
concentration, starting from 1 mm fumarate, but
decreases at concentrations higher than 2 mm fuma-
rate, due to inhibition of NadB by fumarate [9]. There-
fore, to evaluate quinolinate formation, the assay
routinely used contained 70 lg of NadA, 30 lgof
NadB and 1 mm fumarate under anaerobic conditions.
AB
Fig. 1. Production and purification of NadA from Bacillus subtilis.
(A) 11% SDS ⁄ PAGE of NadA–His before and after purification in a
glove box. Std, molecular markers; P, pellet; S, soluble fraction;
NadA–His, purified protein. (B) Visible absorption spectrum of
NadA–His purified under anaerobic conditions (
_______
) and after 2 h
of exposure to air (- - -).
NadA and NadB from B. subtilis I. Marinoni et al.
5092 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
An apparent K

m
of 0.36 ± 0.05 mm was calculated for
DHAP at 25 °C, using oxygen as electron acceptor.
The specific activity of NadA from B. subtilis was
0.05 ± 0.01 lmolÆmin
)1
Æmg
)1
in the presence of fuma-
rate as electron acceptor for NadB, and 0.027 ±
0.01 lmolÆmin
)1
Æmg
)1
using oxygen to reoxidize
NadB. These values were more than two times
higher than that reported for NadA from E. coli
(0.015 lmolÆmin
)1
Æmg
)1
using fumarate) [20,21],
and comparable to the results described for SufE3
purified from A. thaliana, which catalyzes the for-
mation of quinolinate with a specific activity of
0.05 lmolÆmin
)1
Æmg
)1
using fumarate as electron

acceptor for NadB [7]. Similar data were obtained if
NadA without tags or GST–NadA was used in the
assay mixture instead of NadA–His, ruling out
the possibility that the presence of a tag at either the
N-terminus or C-terminus had any effect on the
enzymatic activity.
NadA from B. subtilis contains an oxygen-labile
[4Fe–4S]
2+
cluster as a cofactor
Figure 1B shows the absorbance spectrum of NadA
from B. subtilis purified under anaerobic conditions.
The shoulder at 420 nm in the spectrum suggests the
presence of an [Fe–S] cluster in the protein. This clus-
ter appears to be oxygen-sensitive, because absorption
in the visible region was altered after exposure to air,
with a progressive decrease of the absorption in the
420 nm region (Fig. 1B). Using the protein purified in
the glove box, it was possible to determine that the
protein contained 3.8 ± 0.2 mol iron ⁄ mol NadA and
3.3 ± 0.01 mol inorganic sulfide ⁄ mol protein, suggest-
ing the presence of one [4Fe–4S]
2+
cluster per mono-
mer of NadA. In accordance with this finding,
complete loss of activity was detected for NadA from
B. subtilis purified under anaerobic conditions and
exposed to oxygen overnight, as the cluster integrity is
compromised in such conditions. These results are in
agreement with the data reported for NadA from

E. coli and A. thaliana, which contain one highly oxy-
gen-sensitive [4Fe–4S]
2+
cluster per monomer of NadA
[7,20,21]. The data are in keeping with the hypothesis
proposed by Sun & Setlow [24], who suggested that
NadA from B. subtilis may contain an [Fe–S] cluster,
on the basis of the observation that, like E. coli,
B. subtilis iscS
)
strains are auxotrophic for nicotinic
acid and are unable to synthesize NAD de novo.
Further characterization of the [Fe–S] cofactor was
performed by Mo
¨
ssbauer and EPR spectroscopy
(Figs 2 and 3, respectively). The Mo
¨
ssbauer spectrum
of an NadA sample, recorded at 77 K, is shown in
Fig. 2A. At first glance, it is appropriate to fit this
spectrum with one quadrupole doublet, representing
100% of the iron in the NadA sample. The resulting
fit parameters (isomer shift d = 0.44 mmÆs
)1
, quadru-
pole splitting DE
Q
= 1.05 mmÆs
)1

, and line width
G = 0.48 mmÆs
)1
) are characteristic for [4Fe–4S]
2+
clusters. The [4Fe–4S]
2+
clusters in other biological
systems exhibit similar Mo
¨
ssbauer parameters
[21,25,26]. The Mo
¨
ssbauer spectrum of NadA that was
exposed to air at room temperature (for 30 min), mea-
sured at 77 K, reveals that the [4Fe–4S]
2+
clusters are
oxygen-sensitive and are decomposed (Fig. 2B).
About 55% of the iron in that spectrum still repre-
sents [4Fe–4S]
2+
clusters; the remaining 45% of
the absorption pattern appears as a quadrupole
doublet with Mo
¨
ssbauer parameters (d = 0.27 mmÆs
)1
,
DE

Q
= 0.53 mmÆs
)1
and G = 0.35 mm Æs
)1
) that are
A
B
C
Fig. 2. NadA contains a [4Fe–4S] cluster: Mo
¨
ssbauer spectra mea-
sured at 77 K. (A) The quadrupole doublet represents [4Fe–4S]
2+
clusters. (B) NadA exposed to air for 30 min. The two quadrupole
doublets represent [4Fe–4S]
2+
clusters (dashed line) and, in addi-
tion, high-spin (S =5⁄ 2) tetrahedral-sulfur-coordinated iron sites
(dotted line) (see text). (C) Reanalysis of the measured spectrum
from (A) with two quadrupole doublets representing the 3 : 1 bind-
ing motif of the [4Fe–4S]
2+
clusters (see text). The solid line is the
envelope of the dashed and dotted lines in (B) and (C).
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5093
characteristic for high-spin (S =5⁄ 2) tetrahedral-sul-
fur-coordinated iron sites as observed in [2Fe–2S]
2+

clusters [21,27]. Oxygen sensitivity has been observed
for [4Fe–4S]
2+
clusters in other proteins, where this
sensitivity leads to partial or total degradation of these
clusters. Such proteins have in common that their
[4Fe–4S]
2+
clusters are ligated by only three cysteines,
whereas the fourth iron is coordinated by a nonprotein
ligand [28,29]. It was thus tempting to reanalyze the
Mo
¨
ssbauer spectrum of NadA (which was not exposed
to air), but now assuming two different kinds of iron
sites in the [4Fe–4S]
2+
cluster, which is coordinated
with a 3 : 1 ratio of cysteine to noncysteine. This situa-
tion requires two quadrupole doublets with an area
ratio of 3 : 1, instead of one doublet only. A tetrahe-
dral-coordinated Fe
2.5+
site, with the cysteine ligand
replaced by a nonsulfur ligand, i.e. nitrogen or oxygen,
is expected to exhibit an increase of isomer shift by
around 0.05–0.1 mmÆs
)1
in comparison with the three
tetrahedral-sulfur-coordinated Fe

2.5+
sites. Visualiza-
tion of this specific 3 : 1 binding motif in a Mo
¨
ssbauer
spectrum was provided before for the [4Fe–4S]
2+
clus-
ters in the ferredoxin of the anaerobic ribonucleotide
reductase from E. coli [30], in the ferredoxin from the
hyperthermophilic archeon Pyrococcus furiosus [31], in
the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate syn-
thase from A. thaliana [25], and in the radical S-adeno-
sylmethionine enzyme coproporphyrinogen III oxidase
HemN [26]. A corresponding fit of the Mo
¨
ssbauer
spectrum of NadA using two quadrupole doublets
(Fig. 2C) yields the following: doublet I (dashed
line; the relative absorption area 75% was fixed
in the fit; d
I
= 0.42 mmÆs
)1
, DE
QI
= 1.05 mmÆs
)1
,
G

I
= 0.43 mmÆs
)1
) represents tetrahedral-sulfur-coordi-
nated Fe
2.5+
sites, and doublet II (dotted line; the rela-
tive area 25% was fixed in the fit; d
II
= 0.52 mmÆs
)1
,
DE
QII
= 1.09 mmÆs
)1
, G
II
= 0.42 mmÆs
)1
) represents
the tetrahedral-coordinated Fe
2.5+
site with the cyste-
ine ligand replaced by a noncysteine ligand. The
Mo
¨
ssbauer parameters of the two doublets are in rea-
sonable agreement with those reported for [4Fe–4S]
2+

clusters in other proteins with this specific 3 : 1 bind-
ing motif [25,26,30,31].
The EPR spectrum of the ‘as isolated’ protein (not
presented) showed a trace contribution from a
[3Fe–4S] center at g = 2.031 and a relatively minor
amount of free iron at g = 4.2. However, the spec-
trum did contain a relatively significant contribution
from Cu
2+
, so the ‘as isolated’ spectrum was sub-
tracted from the spectra of the reduced samples to pre-
vent this baseline signal distorting the EPR spectra of
the [Fe–S] center at low fields. The difference
(reduced ) oxidized) EPR spectra of the NadA protein
reduced at pH 8 and pH 10 are presented in Fig. 3.
The sharp derivative signal around g = 2.00 arises
from the radical of methyl viologen, which was added
to the samples as a redox mediator. The EPR spec-
trum of the reduced NadA protein produces an EPR
spectrum in the pH 10 sample at 15 K, which is typical
of a [4Fe–4S]
1+
center with g
1
= 2.054 and
g
2,3
= 1.932. Interestingly, the sample at pH 8 indi-
cates that the [Fe–S] center exists in two slightly differ-
ent forms, with this difference being indicated by a

split in the high field feature with features at g = 1.94
and g = 1.89. This could be caused by slight differ-
ences in folding of the protein, or a charged residue
close to the [Fe–S] center that has a pK
a
close to that
of the sample at pH 8 so that it is only charged in a
fraction of the samples (about 50%). The shift to
pH 10 clearly favors the g = 1.93 ⁄ g = 1.94 fea-
ture ⁄ conformation ⁄ state. Given that it is thought from
studies reported in this article that the fourth ligand to
this [4Fe–4S]
2+
center is not a cysteine, it is tempting
to speculate that the two different forms of the [Fe–S]
cluster detected at pH 8 may reflect differences in the
fourth noncysteine ligand. We estimate that 70–80%
of the maximal [Fe–S] content of these samples is
contributing to the EPR spectra recorded.
Identification of [Fe–S] cluster-binding residues
of NadA
Recent studies on NadA from E. coli and on SufE3
from A. thaliana demonstrated that these enzymes
harbor a [4Fe–4S]
2+
cluster that is essential for the
A
B
Fig. 3. NadA contains a [4Fe–4S] cluster: EPR spectra of NadA
reduced at pH 8 and pH 10. NadA was reduced with sodium dithio-

nite and methyl viologen, as described in Experimental procedures.
The two spectra presented represent the difference between the
reduced sample and the unreduced control. The experimental con-
ditions for acquisition of the spectrum were: microwave power,
2 mW; modulation amplitude, 0.1 mT; temperature, 15 K.
NadA and NadB from B. subtilis I. Marinoni et al.
5094 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
activity [20,21], but no information is available regard-
ing the residues involved in binding of the [Fe–S] clus-
ter. The canonical binding motif for the [4Fe–4S]
2+
cluster CXXCXXCX was found in many quinolinate
synthases from bacteria (including E. coli and Synecho-
cystis), and it was proposed that the cysteines of this
pattern are involved in the binding of the cluster.
However, as shown in Fig. 4, NadA from B. subtilis
lacks this typical cysteine motif and shows a different
arrangement of conserved cysteines from that in E. coli
NadA. The same observation was reported for the
homologous sequence of the bacterial quinolinate
synthase found in chloroplasts of A. thaliana,
Oryza sativa, poplar, medicago and other plant species,
and for the enzyme from P. horikoshii, an anaerobic
hyperthermophilic archaeon whose crystal structure
was solved in 2005 [22]. On the other hand, all the res-
idues involved in the binding of malate in the crystal
structure of NadA from P. horikoshii [22] are strictly
conserved in all the NadA sequences reported so far in
the data banks. Figure 4 reports only few of them, for
reasons of clarity. The sequence alignment analyses

suggest that all quinolinate synthases may share the
unique triangular architecture described for the protein
from P. horikoshii. Unfortunately, this partial structure
lacks the [Fe–S] cluster, and the three surface loops
that contain two highly conserved cysteine residues are
disordered. Therefore, the question of which residues
are important for the binding of the [Fe–S] cluster is
still unanswered. The multiple alignment shows that
three cysteines are strictly conserved in all the plant
and bacterial sequences reported so far, and thus are
very good candidates as iron ligands (Fig. 4). Muta-
genesis studies on NadA from B. subtilis allowed us to
substantiate this hypothesis. B. subtilis nadA encodes
six cysteine residues. Three of them are not shared
with all the proteins represented in Fig. 4 but are well
conserved in the MF_00569 family, one of the three
NadA families of the HAMAP database [32], which
comprises mainly proteins from Gram-positive bacteria
and some archeans of the genus Halobacterium. This
family is very distinct from the other two: the
MF_0567 family (including E. coli NadA), comprising
proteins mainly from Gram-negative bacteria, and the
MF_00568 family, which contains NadA proteins from
bacteria and archeans (e.g. from Mycobacterium tuber-
culosis and P. horikoshii) and plastids. In contrast,
Cys110, Cys230 and Cys320 in NadA from B. subtilis
are strictly conserved in all the NadA sequences
reported so far. Single point mutations to serine were
carried out for all the six residues (Cys82, Cys110,
Cys230, Cys259, Cys318 and Cys320), and the mutant

enzymes purified were tested for enzymatic activity and
iron content (Table 1). In total, six single NadA
mutants and a double mutant carrying the
C318S ⁄ C320S substitution were generated. The yield
and stability of all mutated proteins were comparable
to the those obtained for the wild-type NadA. The
enzymes with mutations of nonconserved residues,
C82S and C259S, showed the same activity, the same
iron content and the same spectral properties as the
wild-type. In contrast, the enzymes with mutations at
conserved residues, C110S and C230S, were almost
colorless and inactive, indicating that these residues
are absolutely vital for [Fe–S] cluster formation
(Table 1). The third residue conserved in all the NadA
sequences is Cys320. The C320S mutant was inactive
but was still able to bind 1.5 iron atoms ⁄ mol protein,
probably because, in the absence of Cys320, Cys318
may play an ancillary role in iron binding. In contrast,
the C318S mutant was fully active and was able to
bind 3.1 iron atoms ⁄ mol protein, unlike the double
mutant C318S ⁄ C320S, which was colorless and inac-
tive. Taken together, the data suggest that in NadA
from B. subtilis the [4Fe–4S]
2+
cluster is coordinated
by three highly conserved cysteine residues (Cys110,
Cys230, and Cys320). The results of site-directed muta-
genesis are in agreement with the fit of the Mo
¨
ssbauer

and EPR spectra reported above, suggesting that
NadA presents a new noncanonical binding motif that,
we propose, may be common to other quinolinate
synthases from different sources.
In vivo Nic phenotype verification of NadA
mutants
As previously reported [24], mutations in the nadBCA
or in the divergent iscS ⁄ nifS operons confer on B. sub-
tilis a Nic
)
phenotype (nicotinic acid requirement in
minimal medium, due to impairment of the de novo
pathway). To verify the phenotype conferred in vivo by
the cysteine to serine substitutions described in the pre-
vious section, we tested the ability to grow in minimal
medium with and without nicotinic acid of the six
B. subtilis NadA single mutants C82S, C110S, C230S,
C259S, C318S, and C320S, and, as a negative control,
of the DnadA mutant obtained with the allelic switch
protocol described in Experimental procedures [33].
The phenotype of isolated clones that each bear a
single cysteine to serine mutation is shown in Fig. 5.
After 24 h of growth in aerobic conditions on minimal
medium with glucose (0.5%) and tryptophan
(50 lgÆmL
)1
), the C110S, C230S, C320S and C318S
mutants showed, in the absence of nicotinic acid, the
same growth impairment as a DnadA strain. The addi-
tion of 0.5–50 lgÆmL

)1
nicotinic acid was sufficient to
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5095
promote normal growth. The C110S, C230S and
C320S mutations thus confer on B. subtilis a Nic
)
phe-
notype, confirming the observations made in vitro with
the purified mutant enzymes. In contrast, the absence
of involvement of Cys318 in NadA activity was not
confirmed by the in vivo study: this cysteine must play
NADA_ECOLI MSVMFDPDTAIYPFPPKPTPLSIDEKAYYREKIKRLLKERNAVMVAHYYTDPEIQQLAEETGGCI SDSLEMARFGAKHP ASTLLVAGVRFMGETAKILSPEK 102
NADA_SALTY MSVMFDPQAAIYPFPPKPTPLNDDEKQFYREKIKRLLKERNAVMVAHYYTDPEIQQLAEETGGCI SDSLEMARFGTKHA ASTLLVAGVRFMGETAKILSPEK 102
NADA_BACSU MSILDVIKQSNDMMPESYKELSRKDMETRVAAIKKKFGSRLFIPGHHYQKDEVIQFADQTG DSLQLAQVAEKNKE ADYIVFCGVHFMAETADMLTSEQ 98
NADA_HELPY MPTDNDLKAAILELLRDLDVLLVAHFYQKDEIVELAHYTG DSLELAKIASQS-D KNLIVFCGVHFMGESVKALAFDK 76
NADA_METTH MLNQLQRDILRLKKEKNAIILAHNYQSREIQEIADFKG DSLELCIEASRIEG KDIVVFCGVDFMAETAYILNPDK 75
NADA_METJA MSMDIVERINKLKEEKNAVILAHNYQPKEIQKIADFLG DSLELCIKAKETD ADIIVFCGVDFMGESAKILNPEK 74
NADA_PYRHO MDLVEEILRLKEERNAIILAHNYQLPEVQDIADFIG DSLELARRATRVD ADVIVFAGVDFMAETAKILNPDK 72
NADA_AQUAE MVQLALKEEKELTKEEIKELQKEVRRLAKEKNAVILAHYYQRPEVQDIADFVG DSLELARKASQTD ADIIVFCGVRFMCETAKIVNPEK 89
NADA_SYNY3 MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG DSLGLSQQAASTD ADVIVFAGVHFMAETAKILNPHK 85
NADA_SYNEC MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG DSLGLSQQAASTD ADVIVFAGVHFMAETAKILNPHK 85
NADA_CYAPA MSIFLKNKQFENITSQEQTKNNYKQLINDIQTLKKDLNAIILAHYYQEPDIQDVADFLG DSLGLAREAAKTN ADIIVFAGVHFMAETAKILNPEK 95
NADA_EHRCR MKELDVIT LLQEIRHLAQESNAVILAHYYQDSEIQDIADFIG DSLELSRKAATTT ADVIVFCGVYFMAEVAKIINPAK 78
NADA_MYCLE MTVLNGMEPLAGDMTVVIAGITDSPVGYAGVDGDEQWATEIRRLTRLRGATVLAHNYQLPAIQDIADYVG DSLALSRIAAEVP EETIVFCGVHFMAETAKILSPNK 106
NADA_MYCTU MTVLNRTDTLVDELT ADITNTPLGYGGVDGDERWAAEIRRLAHLRGATVLAHNYQLPAIQDVADHVG DSLALSRVAAEAP EDTIVFCGVHFMAETAKILSPHK 103
NADA_ATHAL VPSFEPFPSLVLTAHGIEAKGSFAQAQAKYLFPEESRVEELVNVLKEKKIGVVAHFYMDPEVQGVLTAAQKHWPHISISDSLVMADSAVTMAKAGCQFITVLGVDFMSENVRAILDQAGF 120
NADA_OSATI MFLSPNESKTSELVKSLREKKIGIVAHFYMDPEVQGILTASKKHWPHIHISDSLVMADSAVKMAEAGCEYITVLGVDFMSENVRAILDQAGY 92
: .* * : . *** :. . . : . ** ** * . :
NADA_ECOLI TILMPT-LQAEC

SLDLGCPVEEFNAFCDAHPDRT VVVYANTSAAVKARAD WVVTSSIAVELIDHL DSLGEKIIWAPDKHLGRYVQKQTGG 191
NADA_SALTY TILMPT-LAAECSLDLGCPIDEFSAFCDAHPDRT VVVYANTSAAVKARAD WVVTSSIAVELIEHL DSLGEKIIWAPDRHLGNYVQKQTGA 191
NADA_BACSU QTVVLPDMRAGCSMADMADMQQTNRAWKKLQHIFGDTIIPLTYVNSTAEIKAFVGKHG-GATVTSSNAKKVLEWA FTQKKRILFLPDQHLGRNTAYDLGIALEDMAVWDPMKDEL 212
NADA_HELPY QVIMP KLSCCSMARMIDSHYYDRSVHLLKECGVKEFYPITYINSNAEVKAKVAKDD-GVVCTSRNASKIFNHA LKQNKKIFFLPDKCLGENLALENGLKSAILGANS 182
NADA_METTH KILIPD-RGAECPMAHMLSAEDVRMARKRYPDAA VVLYVNTLAEAKAEAD ILCTSANAVRVVES LDEDLVLFGPDRNLAWYVQEHT 160
NADA_METJA KVLMPEIEGTQCPMAHQLPPEIIKKYRELYPEAP LVVYVNTTAETKALAD ITCTSANADRVVNS LDADTVLFGPDNNLAYYVQKRT 160
NADA_PYRHO VVLIPS-REATCAMANMLKVEHILEAKRKYPNAP VVLYVNSTAEAKAYAD VTVTSANAVEVVKK LDSDVVIFGPDKNLAHYVAKMT 157
NADA_AQUAE KVLHPN-PESGCPMADMITAKQVRELREKHPDAE FVAYINTTADVKAEVD ICVTSANAPKIIKK LEAKKIVFLPDQALGNWVAKQV 174
NADA_SYNY3 LVLLPD-LEAGCSLADSCPPREFAEFKQRHPDHL VISYINCTAEIKALSD IICTSSNAVKIVQQ LPPDQKIIFAPDRNLGRYVMEQTGR 173
NADA_SYNEC LVLLPD-LEAGCSLADSCPPREFAEFKQRHPDHL VISYINCTAEIKALSD IICTSSNAVKIVQQ LPPDQKIIFAPDRNLGRYVMEQTGR 173
NADA_CYAPA MVLLPD-LNAGCSLADSCPPEIFSEFKKAHSDHL VISYINCSASIKAMSD IICTSANAVDIVNK IPLTQPILFAPDQNLGRYVISKTGR 183
NADA_EHRCR KVLLPD-LNAGCSLADSCDAESFKKFRELHKDCV SITYINSLAEVKAYSD IICTSSSAEKIIRQ IPEEKQILFAPDKFLGAFLEKKTNR 166
NADA_MYCLE TVLIPD-QRAGCSLADSITPDELCAWKDEHPGAA VVSYVNTTAEVKALTD ICCTSSNAVDVVES IDPSREVLFCPDQFLGAHVRRVTGRK 195
NADA_MYCTU TVLIPD-QRAGC
SLADSITPDELRAWKDEHPGAV VVSYVNTTAAVKALTD ICCTSSNAVDVVAS IDPDREVLFCPDQFLGAHVRRVTGRK 192
NADA_ATHAL EKVGVYRMSDETIGCSLADAASAPAYLNYLEAASRSPPS LHVVYINTSLETKAFAHELVPTITCTSSNVVQTILQAFAQMPELTVWYGPDSYMGANIVKLFQQMTLMTNEEIANIHPK 238
NADA_OSATI SKVGVYRMSSDQIGCSLADAASSSAYTHFLKEASRSPPS LHVIYINTSLETKAHAHELVPTITCTSSNVVATILQAFAQIPGLNVWYGPDSYMGANIADLFQRMAVMSDEEIAEVHPS 210
*.: : * * ** ** . . : : ** :.
NADA_ECOLI DILCWQGACIVHDEFKTQALTRLQEEYPDAAILVHPES PQAIVDMADAVGSTSQLIAAAK TLPH-QRLIVATDRGIFYKMQQAVPDKE 278
NADA_SALTY DVLCWQGACIVHDEFKTQALTRLKKIYPDAALLVHPES PQSIVEMADAVGSTSQLIKAAK TLPH-RQLIVATDRGIFYKMQQAVPEKE 278
NADA_BACSU V AESGHTNVKVILWKGHCSVHEKFTTKNIHDMRERDPDIQIIVHPEC SHEVVTLSDDNGSTKYIIDTIN QAPAGSKWAIGTEMNLVQRIIHEHPDK- 308
NADA_HELPY QEEIKNADVVCYNGFCSVHQLFKLEDIEFYRQKYPDILIAVHPEC EPSVVSNADFSGSTSQIIEFVE KLSPNQKVAIGTESHLVNRLKAKRHHQ- 276
NADA_METTH DKTIIPIPEEGHCYVHKMFTAGDVMAAKEKYPEAELLIHPEC DPEVQELADHILSTGGMLRRVL ESDA-ESFIIGTEVDMTTRISLESD 248
NADA_METJA DKKVIAIPEGGGCYVHKKFTIDDLKRVKSKYPNAKVLIHPEC SPELQDNADYIASTSGILRIVL ESDD-EEFIIGTEVGMINRLEIELEKL- 250
NADA_PYRHO GKKIIPVPSKGHCYVHQKFTLDDVERAKKLHPNAKLMIHPEC IPEVQEKADIIASTGGMIKRAC EWD EWVVFTEREMVYRLRKLYPQ 244
NADA_AQUAE PEKEFIIWK-GFCPPHFEFTYKELEKLKEMYPDAKVAVHPEC HPRVIELADFVGSTSQILKYAT SVDA-KRVIVVTEVGLKYTLEKINPNKE 264
NADA_SYNY3 EMVLWQGSCIVHETFSERRLLELKTQYPQAEIIAHPEC EKAILRHADFIGSTTALLNYSG KSQG-KEFIVGTEPGIIHQMEKLSPSKQ 260
NADA_SYNEC EMVLWQGSCIVHETFSERRLLELKTQYPQAEIIAHPEC EKAILRHADFIGSTTALLNYSG KSQG-KEFIVGTEPGIIHQMEKLSPSKQ 260
NADA_CYAPA DLLLWPGSCIVHETFSEKKIFEFQSLYPTAEVIAHPEC EPTILKHANYIGSTTSLLQYVK NSKK-TTFIVITEPGIIHQMKKSCPEKQ 270
NADA_EHRCR KMILWPGTCIVHESFSERELIDMMVRHDKAYVLAHPEC PGHLLKYAHFIGSTTQLLKFSS DNPN-SEFIVLTEEGIIHQMKKVSSGST 253

NADA_MYCLE NVYVWMGECHVHAGINGDELVDQARANPDAELFVHPECGCSTSALYLAGEGAFPPDRVKILSTGGMLTAAR QTQY-RKILVATEVGMLYQLRRAAPEID 293
NADA_MYCTU NLHVWAGEC
HVHAGINGDELADQARAHPDAELFVHPECGCATSALYLAGEGAFPAERVKILSTGGMLEAAH TTRA-RQVLVATEVGMLHQLRRAAPEVD 290
NADA_ATHAL HSLDSIKSLLPRLHYFQEGTCIVHHLFGHEVVERIKYMYCDAFLTAHLEVPG EMFSLAMEAKKREMGVVGSTQNILDFIKQKVQEAVDRNVDDHLQFVLGTESGMVTSIVAVIRSLL 355
NADA_OSATI HNKKSINALLPRLHYYQDGNCIVHDMFGHEVVDKIKEQYCDAFLTAHFEVPG EMFSLSMEAKTRGMGVVGSTQNILDFIKNHLMEALDRNIDDHLQFVLGTESGMITSIVAAVRELF 327
* * * : : : * * ** :: : *: : :
NADA_ECOLI LLEAPTAGEG ATCRSCAHCPWMAMNGLQAIAEALEQEGSN HEVHVDERLRERALVPLN 336
NADA_SALTY LLEAPTAGEG ATCRSCAHCPWMAMNGLKAIAEGLEQGGAA HEIQVDAALREGALLPLN 336
NADA_BACSU QIESLN PDMCPCLTMNRIDLPHLLWSLEQIEKGEP SGVIKVPKAIQEDALLALN 362
NADA_HELPY NTFILS STLALCPTMNETTLKDLFEVLKAYKNHRA YNTIELKDEVARLAKLALT 330
NADA_METTH KKTIPL LEEAICENMKLHTLEKVKNSLINEEF VVTVPDEIARRARRAVE 297
NADA_METJA GKKKTLIPL RKDAICHEMKRITLEKIEKCLLEERY EIKLEKEIIEKAQKAIE 302
NADA_PYRHO KKFYPA REDAFCIGMKAITLKNIYESLKDMKY KVEVPEEIARKARKAIE 293
NADA_AQUAE YIFPQSMNY CGTVYCCTMKGITLPKVYETLKNEIN EVTLPKDIIERARRPIE 316
NADA_SYNY3 FIPLPNNSN CDCNECPYMRLNTLEKLYWAMQRRSP EITLPEATMAAALKPIQ 312
NADA_SYNEC FIPLPNNSN CDCNECPYMRLNTLEKLYWAMQRRSP EITLPEATMAAALKPIQ 312
NADA_CYAPA FLALPTVSG CACNECPHMRLNTLEKLYLAMKTRSP QIEIPESILLNAKKPIE 322
NADA_EHRCR FYIVKTSDSG G-CVSCSKCPHMRLNTLEKLYLCLKNGYP EITLDPEISSMAKRSLD 308
NADA_MYCLE FRAVNDRAS CKYMKMITPGALLRCLVEGTD EVHVDSEIAAAGRRSVQ 340
NADA_MYCTU FRAVNDRAS CKYMKMITPAALLRCLVEGAD EVHVDPGIAASGRRSVQ 337
NADA_ATHAL G SSANSKLKVEVVFPVSSDSMTKTSSDSSNSIKVGDVA LPVVPGVAGGEGCSIHGGCASCPYMKMNSLSSLLKVCHKLPDLENVYGGFIAERFKRQTPQGKLIADVGCEPIL 467
NADA_OSATI DSYKTSQQSANIEVEIVFPVSSDAVSNTSVNGSHHLDSSTVTDLDNVSVVPGVSSGEGCSIHGGCASCPYMKMNSLRSLLKVCHQLPDRDNRLVAYQASRFNAKTPLGKLVAEVGCEPIL 447
* * : . .:
NADA_ECOLI RMLDFAATLRG 347
NADA_SALTY RMLDFAATLRA 347
NADA_BACSU RMLSIT 368
NADA_HELPY KMMELS 336
NADA_METTH RMIRVSE 304
NADA_METJA RMLRI 307
NADA_PYRHO RMLEMSK 300

NADA_AQUAE RMLELS 322
NADA_SYNY3 RMLAMS 318
NADA_SYNEC RMLAMS 318
NADA_CYAPA RMLEMSN 329
NADA_EHRCR AMLKMS 314
NADA_MYCLE RMIEIGLPGGGE 352
NADA_MYCTU RMIEIGHPGGGE 349
NADA_ATHAL HMRHFQANKELPDKLVHQVLSCESKR 493
NADA_OSATI HMRHFQATKRLPDKLVHHVIHGKGEPTS 475
* .
NadA and NadB from B. subtilis I. Marinoni et al.
5096 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
a role for NadA in vivo, as its substitution with a
serine conferred a clear Nic
)
phenotype.
Characterization of NadB
NadB cloning, expression and purification were carried
out as described in Experimental procedures. Typically,
about 60 mg of pure enzyme was isolated from
6 L of bacterial growth medium. The homogeneity
of the preparation was confirmed by SDS ⁄ PAGE
and N-terminal sequence analysis (data not shown).
The spectral properties of the flavoenzyme (Fig. 6)
were very similar to those of NadB from E. coli,
although the absorbance maximum was shifted to a
Table 1. Identification of [Fe–S] cluster-binding residues of NadA. All of the cysteine residues were mutated to serine, and the mutants
were probed for iron content and enzymatic activity in a glove box using fumarate as electron acceptor for NadB. A double mutant
(C318S ⁄ C320S) was also obtained and tested. +, ability to grow in minimal medium without nicotinic acid; ), requirement for
0.5–50 mgÆmL

)1
nicotinic acid for growth in minimal medium. ND, not determined.
Enzyme
Iron content ⁄
mol protein
Enzymatic activity under
anaerobic conditions (UÆmg
)1
)
Nic phenotype
of B. subtilis
Wild-type 3.8 ± 0.3 0.050 ± 0.008 +
C82S 4.3 ± 0.5 0.048 ± 0.010 +
C110S 0.4 ± 0.1 No activity )
C230S 0.6 ± 0.2 No activity )
C259S 4.5 ± 0.5 0.038 ± 0.007 +
C318S 3.3 ± 0.6 0.033 ± 0.005 )
C320S 1.5 ± 0.3 No activity )
C318S ⁄ C320S 0.3 ± 0.2 No activity ND
Fig. 5. In vivo Nic phenotype verification of
NadA mutants. Growth after 24 h at 37 °C
of the wild-type (WT) (PB168, trpC
2
) and
mutated derivative B. subtilis strains,
obtained by allelic switch. The strains were
streaked on minimal Davis and Mingioli agar
medium [43] in the presence of 0.5%
glucose, 50 lgÆmL
)1

tryptophan and, where
indicated, of nicotinic acid (Nic, 50 lgÆmL
)1
).
Fig. 4. Multiple alignment of NadA primary sequences from different bacteria and plants. Conserved cysteines are indicated in bold and
labeled with arrows. All the cysteines of Bacillus subtilis, mutated to serine in the present work, are shaded in gray. The residues involved
in the binding of malate in NadA from Pyrococcus horikoshii are indicated in bold. NADA_ECOLI: from Escherichia coli (Swiss Prot accession
number P11458). NADA_SALTY: from Salmonella typhimurium (Swiss Prot accession number P24519). NADA_BACSU: from B. subtilis
(Swiss Prot accession number O32063). NADA_HELPY: from Helicobacter pylori (Swiss Prot accession number O25910). NADA_METTH:
from Methanobacterium thermoautotrophicum (Swiss Prot accession number O27855). NADA_METJA: from Methanococcus jannaschii
(Swiss Prot accession number Q57850). NADA_PYRHO: from P. horikoshii (Swiss Prot accession number O57767). NADA_AQUAE: from
Aquifex aeolicus (Swiss Prot accession number O67730). NADA_SYNY3: from Synechocystis sp. strain PCC 6803 (Swiss Prot accession
number P74578). NADA_SYNEC: from Synechocystis (GenBank accession number NP_442873). NADA_CYAPA: from Cyanophora paradoxa
(Swiss Prot accession number P31179). NADA_EHRCR: from Ehrlichia chaffeensis (Swiss Prot accession number O05104). NADA_MYCLE:
from Mycobacterium leprae (Swiss Prot accession number Q49622). NADA_MYCTU: from Mycobacterium tuberculosis (Swiss Prot acces-
sion number O06596). NADA_ATHAL: from Arabidopsis thaliana (GenBank accession number NP_199832). NADA_OSATI: from Oryza sativa
(GenBank accession number ABA_97161).
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5097
lower wavelength (444 nm instead of 452 nm). A rec-
onstitutable apoprotein was obtained and was utilized
to determine the dissociation constant for the FAD–
enzyme complex by the ultrafiltration method, both
in the presence and in the absence of 10 mm fuma-
rate [9], giving values of 4.46 ± 0.5 lm for the free
enzyme and 1.6 ± 0.5 lm for the complex with
fumarate. These results were very similar to the data
described for the enzyme from E. coli (3.8 lm in the
presence of fumarate and 0.6 lm in the absence of
fumarate, respectively [14]), suggesting that, as in the

enzyme from E. coli, in NadB from B. subtilis the
presence of the substrate fumarate in the incubation
mixture does stabilize the holoenzyme significantly.
In contrast, the dissociation constant for the FAD–
enzyme complex did not change if the apoenzyme
was incubated with the coenzyme in the presence of a
stoichiometric amount of NadA, suggesting that this
protein does not influence the binding of FAD to
NadB.
The enzyme from B. subtilis showed typical flavin
fluorescence, with excitation and emission maxima at
450 nm and 520 nm, respectively. The binding of FAD
did not quench the protein fluorescence (excitation at
295 nm, emission at 340 nm) (data not shown). More-
over, upon addition of NadA in NadA ⁄ NadB ratios of
1 : 1 and 2 : 1, the flavin coenzyme fluorescence prop-
erties were still the same as in the absence of NadA.
The aggregation state of pure NadB was determined
by gel filtration. In accordance with the results for the
enzyme from E. coli [9], NadB from B. subtilis is a
dimer of 115 kDa in the absence of NaCl and a mono-
mer of 55 kDa in the presence of 150 mm NaCl. After
incubation with pure NadA in ratios of 1 : 1 or 2 : 1,
under either aerobic or anaerobic conditions, gel filtra-
tion experiments did not show any peaks with a molec-
ular weight equal to the sum of NadA and NadB,
suggesting that the two proteins do not form a stable
complex in such conditions.
The binding of dicarboxylic compounds caused spec-
tral changes similar to those observed in NadB from

E. coli, as shown in Fig. 6A for the binding of fuma-
rate. However, the corresponding dissociation con-
stants were higher for the enzyme from B. subtilis,as
shown in Table 2, which reports the values calculated
for the enzyme from B. subtilis, as well as the corre-
sponding K
d
measured for NadB from E. coli in
50 mm potassium phosphate buffer (pH 8.0) and 20%
glycerol. The opposite was observed for the product
iminoaspartate, which bound more tightly to the
enzyme from B. subtilis (Table 2). In keeping with this
observation, the enzyme showed pronounced product
inhibition when the l-aspartate oxidase activity was
checked at 0.24 mm oxygen and the l-aspartate–fuma-
rate oxidoreductase activity was determined under
anaerobic conditions.
NadB shows three enzymatic activities: l-aspartate–
oxygen oxidoreductase activity, fumarate reductase
activity, and l-aspartate–fumarate oxidoreductase
activity. Regarding the l-aspartate oxidase activity,
using oxygen as electron acceptor, the apparent K
m
(1.0 ± 0.6 mm) and the k
cat
(10.8 ± 1.0 min
)1
) were
calculated, and are reported in Table 3. NadB from
B. subtilis could use fumarate as electron acceptor with

a k
cat
⁄ K
m
ratio comparable to the one reported for the
enzyme from E. coli. As far as the l-aspartate–fuma-
rate oxidoreductase activity was concerned, the double
substrate inhibition pattern described for NadB from
E. coli [14] was also present in the protein from B. sub-
tilis, but in the latter case the substrate inhibition was
greater and the turnover number and the other kinetic
Fig. 6. Purification and spectral properties of NadB from Bacil-
lus subtilis: the visible absorption spectrum of NadB in 50 m
M
potassium phosphate buffer (pH 8.0) containing 20% glycerol, at
25 °C, before (
_______
) and after (- - -) addition of 20 mM fumarate.
Inset: Benesi–Hildebrand plot for the binding of fumarate.
K
d
= 4.4 mM.
Table 2. Dissociation constants for the binding of dicarboxylic com-
pounds to NadB from Bacillus subtilis. The dissociation constants
for dicarboxylic ligands were measured spectrophotometrically by
addition of small volumes of concentrated stock solutions to sam-
ples containing about 10–25 l
M holoenzyme at 25 °Cin50mM
potassium phosphate buffer (pH 8.0) and 20% glycerol. Iminoaspar-
tate was produced by an enzymatic system consisting of

D-aspar-
tate ⁄
D-aspartate oxidase to produce iminoaspartate in situ free of
excess reagents, using a concentration of
D-aspartate of 300 lM.
The corresponding values for the enzyme from Escherichia coli
were evaluated under the same conditions for comparison.
Ligands B. subtilis NadB E. coli NadB
Fumarate 4.4 ± 0.5 m
M 1.14 ± 0.50 mM
Succinate 55 ± 3 mM 2.7 ± 0.7 mM
D
-Aspartate 32 ± 2 mM 9±1mM
Oxaloacetic acid 0.5 ± 0.3 mM 1.7 ± 0.2 mM
Iminoaspartate 0.32 ± 0.10 lM 1.0 ± 0.5 lM
NadA and NadB from B. subtilis I. Marinoni et al.
5098 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
parameters could not be accurately determined, as it
was impossible to work with high l-aspartate or fuma-
rate concentrations. However, taken together, the data
suggest that in the case of NadB from B. subtilis,
fumarate can replace oxygen as electron acceptor, simi-
larly to what has been described for the enzyme from
E. coli, and that the two proteins present very similar
biochemical properties.
NadA–NadB interaction
As reported above, pure and active NadA and NadB
from B. subtilis were obtained in solution in reasonable
amounts, opening the possibility for an investigation
of the complex between NadA and NadB. Such a

multienzymatic complex (sometimes referred to as the
‘quinolinate synthase complex’) has never been
observed, but has been proposed on the grounds of
the following indirect observations: (a) iminoaspartate,
the product of NadB and substrate of NadA, is unsta-
ble in solution, and consequently it is unlikely that it
has to reach NadA simply by diffusing through the
cell; and (b) partial copurification of the two wild-type
enzymes has been obtained in E. coli [34]. On the other
hand, it has been reported that NadA from E. coli can
form quinolinate using iminoaspartate produced by
d-aspartate oxidase [2] or chemically generated in the
assay mixture [35]. Moreover, in T. maritima, NadB is
substituted by an NADP-dependent l-aspartate dehy-
drogenase to produce iminoaspartate [17]. In an
attempt to solve this issue, we utilized the following
different approaches.
The existence of species-specific interactions between
NadA and NadB in quinolinate formation was investi-
gated by evaluating the enzymatic activity of NadA
from B. subtilis in the presence of 20 lg of NadB from
E. coli, using either fumarate or oxygen as electron
acceptor for NadB. The specific activity was
0.04 ± 0.02 lmolÆmin
)1
mg
)1
in the presence of fuma-
rate and 0.027 ± 0.01 lmolÆmin
)1

Æmg
)1
using oxygen.
If compared with the results obtained using NadB
from B. subtilis reported above, these data suggest that
NadA is unable to discriminate between NadB from
B. subtilis and from E. coli, and that the interaction
between the two proteins is not species-specific in this
case.
As the presence of His-tags or GST-tags does not
affect the activity and properties of NadA, it was pos-
sible to apply an affinity capture protocol using recom-
binant GST–NadA or NadA–His. NadA fused to GST
and bound to glutathione–Sepharose (1 mL of resin
saturated with NadA) was incubated in batches with:
(a) pure NadB from B. subtilis (NadA ⁄ NadB ratio
1 : 1 or 2 : 1); (b) a homogenate obtained from E. coli
cells overexpressing NadB from B. subtilis; or (c) a
homogenate of B. subtilis cells (500 lg of total
proteins). The incubation took place under anaerobic
conditions at room temperature for up to 30 min in:
(a) 50 mm Tris ⁄ HCl (pH 7.5) and 0.15 m NaCl; or (b)
50 mm Tris ⁄ HCl (pH 8.0) and 10 mm b-mercaptoetha-
nol. A control experiment was set up by incubating
pure NadB with glutathione–Sepharose without NadA
in order to rule out the possibility of unspecific binding
of NadB to the resin. After extensive washing, the pro-
teins were eluted and subjected to 11% SDS ⁄ PAGE.
The gels were either stained by silver or Coomassie
blue or electroblotted onto a poly(vinylidene difluo-

ride) membrane for N-terminal sequence analysis. A
band corresponding to NadB could be detected in the
samples obtained from the incubation with pure NadB
and with the homogenate of E. coli overexpressing
NadB from B. subtilis. Moreover, the comparison car-
ried out with the control showed that this band was
not due to unspecific binding of NadB to the resin.
The same data were obtained if pure NadB was incub-
ated with NadA–His bound to an Ni
2+
–nitrilotriacetic
acid resin, suggesting that the binding is not dependent
on the presence of a tag either at the N-terminus or at
Table 3. Kinetic parameters for the three activities of NadB. The activity assays were carried out as described in Experimental procedures.
The corresponding values for the enzyme from Escherichia coli are reported in Tedeschi et al. [9,14].
Activity
NadB from B. subtilis NadB from E. coli
K
m
L
–Asp
(mM)
K
m fumarate
(mM) k
cat
(s
)1
)
k

cat
⁄
K
m
L
-Asp
(s
)1
M
)1
)
k
cat
⁄
K
m fumarate
(s
)1
M
)1
)
K
m
L
–Asp
(mM)
K
m fumarate
(mM)
k

cat
(s
)1
)
k
cat
⁄
Km
L
-Asp
(s
)1
M
)1
)
k
cat
⁄
Km
fumarate
(s
)1
M
)1
)
L-Aspartate–oxygen
oxidoreductase
1.0 ± 0.5 0.18 ± 0.02 180 1.74 0.465 267.23
Fumarate reductase 1.6 ± 0.6 15.4 ± 0.8 9625 0.048 0.27 5625
L-Aspartate–fumarate

oxidoreductase
20.0 ± 3 1.43 ± 0.9 0.50 ± 0.3 25 350 2.70 2.50 5.55 2055.55 2220
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5099
the C-terminus of NadA (data not shown). Changing
the incubation buffer, the condition with regard to aer-
obic or anaerobic status, incubation temperature and
time as well as adding 10 mml-aspartate or fumarate
to the incubation mixture did not affect the results. On
the other hand, we were unable to detect any binding
upon incubation with a homogenate of B. subtilis cells
in which NadB expression was induced at the physio-
logical level, probably because NadB possibly bound
to NadA was under the detection limit of the silver
staining. Data comparable to those obtained with
NadB from B. subtilis were obtained when the experi-
ments were repeated using NadB from E. coli, ruling
out the possibility that there is a species-specific inter-
action between NadA and NadB, in accordance with
the results described above obtained by measuring the
enzymatic activity.
As a further step in the study, the ability of NadA
to bind the product iminoaspartate released by B. sub-
tilis NadB was investigated under both aerobic and
anaerobic conditions. Oxaloacetate binds to NadB
with a K
d
of 0.5 mm (Table 2). The addition of ammo-
nium sulfate to the oxaloacetate–NadB complex
resulted in a further spectral perturbation due to the

formation of iminoaspartate, as previously reported
for the enzyme from E. coli [9]. Figure 7 shows the
spectrum of B. subtilis NadB upon binding of
iminoaspartate. Gel filtration on a PD10 column
caused the dissociation of oxaloacetate, but not of
iminoaspartate. The iminoaspartate itself was stabilized
upon binding to NadB, as the observed rate constant of
hydrolysis of free iminoaspartate to oxaloacetate and
ammonia under these conditions was equal to
2.8 ± 0.2 · 10
)1
min
)1
, whereas the apparent rate of
decay of the NadB–iminoaspartate complex to free
holoenzyme was 7.0 ± 0.1 · 10
)3
min
)1
. When the
experiment was carried out in the presence of NadA,
with a 1 : 1 NadA ⁄ NadB ratio, under strictly anaero-
bic conditions, a significant increase in the apparent
rate of decay for the NadB–iminoaspartate complex
(0.027 ± 0.03 min
)1
) was observed (Fig. 7, inset),
suggesting that NadA may perturb the equilibrium of
dissociation by binding to the product iminoaspartate.
In contrast, the kinetic parameters were unaffected by

the presence of NadA when the experiments were
carried out under aerobic conditions, suggesting that
the integrity of the [Fe–S] cluster is important for the
binding of iminoaspartate to NadA.
Conclusions
In the past few years there has been a revival of inter-
est in the study of the biochemistry of NAD, with the
discovery that, in addition to its well-established role
in redox biochemistry, NAD can function as signaling
molecule in a variety of cellular processes [36]. These
multiple functions of NAD imply that its cellular con-
centration is tightly regulated and adjusted to cellular
needs. In particular, NAD homeostasis is maintained
by de novo synthesis or the recycling pathway. Interest
in the de novo biosynthesis of NAD has been further
increased by the discovery that the enzymes involved
in the pathway have acquired different specificities
in different organisms, using different precursors and
effectors, resulting in unexpected complexity. In
eubacteria, the first two steps in de novo synthesis are
catalyzed by the NadA and NadB proteins, which
belong to a large family of proteins with homologs
in bacteria, archaeans and plants. Despite the impor-
tant role played by these enzymes in NAD meta-
bolism in bacteria, NadB has been characterized only
from E. coli and S. tokodaii, and the biochemical
and structural properties of NadA are still largely
unknown.
The experimental findings on NadB from B. subtilis
reported above strongly suggest that the major

biochemical peculiarities of the enzyme are strictly
conserved: spectral properties, aggregation state, bind-
ing of inhibitors and substrate analogs and enzymatic
activity are comparable with those of the enzyme from
E. coli, suggesting that the two proteins share the same
specificities, including the ability to use fumarate as
electron acceptor instead of oxygen. In keeping with
these data, a comparison at the primary structure level
shows that all the residues involved in FAD and
Fig. 7. NadA–NadB interaction. Decay of the NadB–iminoaspartate
complex in the presence of NadA with an NadA ⁄ NadB ratio of
1 : 1, under strictly anaerobic conditions. The visible absorption
spectrum of the NadB–iminoasparate complex prepared as
described in Experimental procedures is shown as a dashed line.
The solid line indicates the spectrum at the end of the process of
decay of the NadB–iminoaspartate complex. Inset: Measurement
of the A
495 nm
was started immediately after removing the
excess reagent by gel filtration. The line is a computer fit of the
first-order kinetics assuming an apparent rate of decay equal to
0.027 min
)1
.
NadA and NadB from B. subtilis I. Marinoni et al.
5100 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
substrate binding are strictly conserved. Moreover, the
availability of active NadA in solution allowed, for
the first time, the investigation of its possible effect on
the properties of NadB, and of whether a complex

between NadA and NadB exists in vitro. The results
show that the biochemical properties of NadB
are unaffected by the presence of NadA in an
NadA ⁄ NadB ratio 1 : 1 or 2 : 1, either under aerobic
or under anaerobic conditions, and that the interaction
between the two enzymes is not species-specific, in
keeping with previous observations reporting the
formation of quinolinate by NadA starting from
iminoaspartate produced by d-aspartate oxidase [2],
l-aspartate dehydrogenase [17], or chemically [35].
Although not species-specific, the binding between
NadA and NadB can be observed by an affinity
capture approach if GST–NadA or NadA–His are
incubated with pure NadB or a homogenate of E. coli
overexpressing NadB. Unfortunately, this interaction is
not tight enough to allow the determination of the cor-
responding K
d
. Moreover, NadA increases the appar-
ent rate of decay of the NadB–iminoaspartate complex
under strictly anaerobic conditions . Taken together,
the data suggest that the two proteins are not tightly
bound to one another and may function as a reversible
multienzyme complex.
The second enzyme in this pathway, NadA, has been
shown to be an iron–sulfur protein in B. subtilis.It
binds a [4Fe–4S]
2+
cluster, which is highly unstable
under aerobic conditions, although the protein in

B. subtilis lacks the previously reported canonical bind-
ing motif for such a cluster. One of the major findings
concerns the identification of the cysteine residues
involved in the binding of the cluster. The site-directed
mutagenesis experiments clearly demonstrate that three
cysteines are essential for this binding: Cys110,
Cys230, and Cys320. Remarkably, together with the
residues involved in the binding of malate in the crys-
tal structure of NadA from P. horikoshii [22], these are
the only residues that are strictly conserved in all the
NadA sequences reported so far, suggesting that the
quinolinate synthases may share the same structural
architecture and a new noncanonical binding for the
[4Fe–4S]
2+
cluster. It has been previously proposed
that NadA may catalyze the formation of quinolinate
through a dehydration mechanism similar to the one
proposed for the [Fe–S]-containing enzymes within the
hydrolyase family. In that case, the [4Fe–4S]
2+
cluster
is bound by three cysteines, whereas the fourth iron is
coordinated by a nonprotein ligand, frequently the
hydroxyl group of the substrate or reaction intermedi-
ate [37]. Therefore, it is possible that the same arrange-
ment exists in NadA.
The essential role played by Cys110, Cys230 and
Cys320 in NadA from B. subtilis was confirmed by the
growth defect shown by the NadA mutants in minimal

medium. The data clearly show that C110S, C230S
and C320S substitutions confer on B. subtilis a Nic
)
phenotype, in keeping with the results from site-direc-
ted mutagenesis studies and Mo
¨
ssbauer spectroscopy.
A considerable discrepancy has been observed between
the in vivo phenotype of the B. subtilis mutant C318S,
which requires nicotinic acid, and the behavior of the
corresponding purified mutant enzyme, which is fully
active when assayed in vitro. It should be pointed out
that in vitro all the enzymes were overexpressed in the
heterologous system of E. coli. On the other hand,
in vivo, the mutant enzymes were expressed at physio-
logical levels and in the natural background. The data
reported above show that Cys318 plays an essential
role in vivo in de novo NAD synthesis, in keeping with
the observation that this cysteine, even if it is not fully
conserved in all the NadA sequences reported in the
data banks, is mainly conserved in the family compris-
ing NadA from the Firmicutes and other Gram-
positive organisms such as Streptomyces spp.
Further studies are in progress in order to address this
particular issue.
Experimental procedures
NadA cloning, protein expression and purification
To clone a GST-tagged NadA, a DNA fragment compris-
ing the nadA coding sequence was produced by amplifica-
tion of chromosomal DNA from PB168 using primers

NadAGEX (EcoRI site) and XNadA (XhoI site, Table S1);
after digestion with EcoRI and Xhol, the fragment was
inserted in the same site of pGEX6p.1 (GE Healthcare, Pis-
cataway, NJ, USA) with the GST at the N-terminal end.
Escherichia coli BL21 cells were transformed with
pGEX6p.1–NadA and inoculated overnight at 37 °CinLB
broth plus 1 mm Fe(III)citrate and 100 lgÆmL
)1
ampicillin,
and the cells were then diluted 1 : 100 and induced over-
night at 28 °C with 1 mm isopropyl thio-b-d-galactoside
(IPTG) at a D
600nm
of 0.6. Before being harvested, cells
were left in a glove box (Iteco, Ravenna, Italy) for 1 h and
then centrifuged at 4400 g (Beckman Coulter, Fullerton
CA, USA) for 15 min at 4 °C. Each step of NadA purifica-
tion was performed under anaerobic conditions in the glove
box. The cells were resuspended in 50 mm Tris ⁄ HCl
(pH 8.0), 300 mm NaCl, 0.5 mgÆmL
)1
lysozyme,
0.1 mgÆmL
)1
RNase, 0.1 mgÆmL
)1
DNase and 1 mm phen-
ylmethanesulfonyl fluoride, and broken by vortex with glass
beads (0.1–0.2 nm; Permax, Milano, Italy). The homoge-
nate was then centrifuged at 12 000 g for 45 min at 4 °C,

and the supernatant was loaded on 6 mL of glutathione–
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5101
Sepharose (GE Healthcare). The column was washed
twice with 50 mm Tris ⁄ HCl (pH 7.5), 300 mm NaCl and
1mm phenylmethanesulfonyl fluoride. GST–NadA bound
to the glutathione–Sepharose was incubated with 80 lLof
Prescission Protease (2 UÆmL
)1
) (GE Healthcare) overnight
at room temperature in 1 mL of 50 mm Tris ⁄ HCl (pH 8.0)
and 300 mm NaCl. After cleavage, NadA was eluted with
seven amino acids of GST left at the N-terminal end
(Gly-Pro-Leu-Gly-Ser-Pro-Glu). Alternatively, GST–NadA
was obtained by eluting with 10 mm glutathione in
50 mm Tris ⁄ HCl (pH 8.0).
To clone the nadA coding sequence gene in pET-28a
(Novagen, Milano, Italy) with the His-tag at C-terminus or
N-terminus, a DNA fragment from PB168 was amplified
with primers (Table S1) NNadA (NdeI) and XNadA (XhoI)
(His-tag at the N-terminal end) and with primers AXho
(XhoI) and ANco (NcoI) (in this primer, a serine was
substituted by a valine) (His-tag at the C-terminal end).
The procedure for overexpression and purification of both
recombinant proteins was the same. E. coli BL21(DE3) cells
were transformed with the plasmid and inoculated in LB
broth plus 1 mm Fe(III) citrate and 30 lgÆmL
)1
kanamycin.
On the following day, cells were diluted 1 : 100 and, at a

D
600nm
of 0.6, were induced overnight with 1 mm IPTG at
28 °C. Cells were left in a glove box for 1 h, and then
harvested by centrifugation at 5000 r.p.m. for 15 min
at 4 °C, resuspended in the glove box in 50 mm
Tris ⁄ HCl (pH 8.0), 1 mm phenylmethanesulfonyl fluoride,
0.1 mgÆmL
)1
RNase, 0.1 mgÆmL
)1
DNase, 0.5 mgÆmL
)1
lysozyme, and 10 mm b-mercaptoethanol, and disrupted by
vortexing with glass beads. The cellular homogenate was
then centrifuged at 12 000 g for 45 min at 4 °C, and the
supernatant was loaded on 6 mL of Ni
2+
–nitrilotriacetic
acid slurry (Qiagen, Milano, Italy) and incubated for
20 min; the column was washed twice with 50 mm
Tris ⁄ HCl, 300 mm NaCl, 25 mm imidazole, 1 mm phenyl-
methanesulfonyl fluoride, and 10 mm b-mercaptoethanol
(pH 8.0), containing 20% glycerol, and the protein was
eluted in 50 mm Tris ⁄ HCl, 300 mm NaCl, 250 mm imidaz-
ole, 1 mm phenylmethanesulfonyl fluoride, 10 mm b-mer-
captoethanol (pH 8.0), and 20% glycerol. The protein was
then loaded onto a PD10 column (GE Healthcare) and
eluted in 50 mm Tris ⁄ HCl (pH 8.0) and 20% glycerol. Pure
protein was stored at )20 °C under strictly anaerobic con-

ditions until use. If needed, buffer change was carried out
in the glove box on a PD10 column.
NadA cloning in pMAD for in vivo allelic
replacement
An NcoI–SalI fragment, comprising the entire nadA gene
and part of the 3¢-end of the nadC coding sequence and the
promoter and 5¢-end of the safA coding sequence, was PCR-
amplified from the B. subtilis 168 (trpC2) wild-type strain
chromosome, using the primers SSafA and N1911 listed in
Table S1, and cloned in the vector pMAD [33], yielding the
vector pMADNAD11. For the construction of the pMADD-
NadA vector for allelic replacement, we cloned in tandem in
pMAD two fragments, an NcoI–EcoRI fragment containing
the first 319 bp of the coding sequence of the nadA coding
sequence, and an EcoRI–SalI fragment containing the last
445 bp of the coding sequence, in this way creating a deletion
of 479 bp and a new EcoRI restriction site.
The allelic switch protocol described by Arnaud et al.
[33] was used to introduce the cysteine to serine coding
nucleotide substitutions and the DnadA mutation. This pro-
tocol is based on chromosome integration–excision by sin-
gle crossing over of the thermosensitive replicon
pMADNAD11, bearing the different cysteine to serine
alleles. The outcomes expected after selection and screening
of plasmid-cured clones are generally two: clones in which
the desired mutation substituted the wild-type allele, or,
alternatively, clones in which the substitution did not occur.
Clones were screened for the Nic
)
phenotype and verified

by sequencing.
When the mutation did not impair de novo NAD synthe-
sis, as was the case for the C82S and C259S mutants, in four
independent allelic switch selections, 100% of the screened
clones were able to grow on minimal medium without
nicotinic acid (Nic
+
phenotype), and at least one of these
clones, verified by sequencing of the safA¢–nadA–¢nadC
region of its chromosome, bore the expected mutation in the
nadA gene, demonstrating that the mutation did not affect
de novo synthesis. In contrast, if the cysteine to serine substi-
tution affected de novo synthesis, we observed that from
50% to 100% (in the case of gene conversion) of the clones
that were candidates to be mutants, obtained from four
independent allelic switch selections, had a Nic
)
phenotype
(nicotinic acid requirement for normal growth on minimal
medium) that was correlated by sequencing with the pres-
ence of the expected nadA nucleotide substitutions. This was
the case for the allelic switch experiments conducted with
pMADNADA11 bearing the mutations C110S, C230S,
C320S, and C318S. In these cases, the Nic
+
clones did not
bear the expected mutation.
NadA mutagenesis
Site-directed mutagenesis of the nadA coding sequence
cloned in the pET28-a vector expressing NadA–His and

pMADNAD11 was performed by using the method of
mutagenic overlapping primer extension by PCR using Pfu-
Ultra (QuickChange IIXL; Stratagene, La Jolla, CA, USA)
or Max-Pfu (Vivantis, Sha Alam Selangor, Malaysia) in the
presence of 3% dimethylsulfoxide by using the forward and
reverse primers listed in Table S1. All of the mutations
introduced were verified by direct sequencing of the plas-
mids used for the expression or the allelic replacement
and, after the allelic switch, by sequencing by PCR of the
B. subtilis genomic DNA.
NadA and NadB from B. subtilis I. Marinoni et al.
5102 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
Mo¨ ssbauer spectroscopy
For Mo
¨
ssbauer spectroscopy, BL21DE3 cells were trans-
formed with the pET28a–NadA vector and grown at 37 °C
in 6 L of MT medium [15 mm (NH
4
)
2
SO4, 80 mm
K
2
HPO
4
,44mm KH
2
PO
4

,3mm sodium dithionite, and
2.3 mm MgSO
4
) supplemented with 0.0015% thiamine,
0.0024% pantothenic acid, 0.002% acid pyridoxine–HCl,
5% glucose, 14 lm
57
FeCl
3
, and 30 lgÆ mL
)1
kanamycin.
Cell growth and NadA protein purification were carried
out as described above.
Mo
¨
ssbauer spectra of 0.7 mm
57
Fe-enriched NadA were
recorded using a home-made spectrometer in the constant
acceleration mode, and analyzed according to Gu
¨
tlich et al.
[38]. Isomer shifts are given relative to a-Fe at room tem-
perature. The spectra obtained at 77 K were measured in a
continuous flow cryostat (Oxford). Spectra were analyzed
by least-square fits using Lorentzian line shape.
EPR spectroscopy
For EPR spectroscopy, the NadA was prepared in LB
broth plus 1 mm Fe(III) citrate and 30 lgÆmL

)1
kanamycin;
0.56 mm NadA in 50 mm Tris ⁄ HCl, 300 mm NaCl, 250 mm
imidazole (pH 8.0), 10 mm b-mercaptoethanol and 20%
glycerol was placed in a 3 mm internal diameter quartz
EPR tube, and 11.5 mm sodium dithionite and 0.14 mm
methyl viologen were added under anaerobic conditions.
The sample was incubated anaerobically for 15 min and
frozen in liquid nitrogen. A control was performed without
dithionite or methyl viologen. An additional sample was
prepared, and 10% of a 2 m glycine ⁄ KOH solution was
added under anaerobic conditions to shift the pH of the
sample to pH 10. This sample was then reduced by the
addition of sodium dithionite and methyl viologen as
described above. EPR spectra were obtained at X-band
using a Bruker ELEXSYSE500 spectrometer, equipped with
an Oxford Instruments ESR900 liquid helium cryostat.
NadA activity assay
Enzymatic activity was determined by measuring quinoli-
nate formation as previously described for the enzyme from
E. coli, with minor changes [19], using either NadB from
B. subtilis or NadB from E. coli in the assay mixture. The
experiments were carried out both in aerobic conditions
using oxygen as electron acceptor for reduced NadB, or in
the glove box under anaerobic conditions using fumarate
instead of oxygen. Unless otherwise specified, 0.5 mL of
reaction mixture contained 2 mm DHAP, 5 lg of catalase,
10 mm FAD, 10 mml-aspartate, 30 lg of NadB, 70 lgof
NadA and 1 mm fumarate (if the assay was carried out
under anaerobic conditions) in 50 mm Tris ⁄ HCl (pH 8.0) at

25 °C. To calculate the K
m
for DHAP, the concentration
was varied in the range 0.1–5 mm. After 20 min of incuba-
tion, the reaction was stopped by adding acetic acid, and
the amount of quinolinate was evaluated by McDaniel’s
method [19].
Iron and sulfide determination
Iron content was determined according to Vanoni et al.
[39]. In detail, 0.3 mL aliquots were denatured following
addition of trichloroacetic acid (10%) and ascorbic acid
(7.5 mm) by heating at 100 °C for 10 min. Precipitated pro-
tein was removed by centrifugation at 10 000 g for 5 min,
and the iron content was determined on aliquots of the
supernatant after correction for dilution. The iron-contain-
ing solution was incubated with 200 lLof75mm ascorbic
acid and 20 lLof10mm 4,4¢-[3-(2-pyridinyl)-1,2,4-tri-
azine5,6-diyl]bis(benzenesulfonic acid) (ferrozine), in a final
volume of 420 lL for 5–10 min. Saturated ammonium ace-
tate (380 lL) was then added to neutralize the solution and
to obtain full development of the color. The absorbance of
the solution at 562 nm was measured, and an extinction
coefficient of 28 mm
)1
Æcm
)1
was used to calculate the iron
concentration. Control experiments were carried out using
an iron solution (iron atomic absorption standard; Sigma,
St Louis MO, USA) as an external standard [39].

The acid-labile sulfur content was determined as
described by Rabinowitz et al. [40].
NadB cloning, overexpression and purification
A DNA fragment comprising the nadB gene sequence was
produced by amplification of chromosomal DNA from
PB168 using primers NNadB (NcoI) and ENadB (EcoRI),
listed in Table S1. After digestion, the fragment was
inserted in the vector pET-Duet (Novagen, Milano, Italy).
E. coli BL21DE3 cells were transformed with the vector
Pet-Duet (NadB) and grown in LB medium at 37 °C plus
100 lgÆmL
)1
ampicillin to a D
600nm
of 0.6. Expression was
induced for 5 h with 1 mm IPTG at 28 °C. Cells were
harvested by centrifugation at 3000 g for 5 min at 4 °C,
and the cellular pellet was stored at )20 °C.
The protein was purified with a procedure that was
simplified in comparison to the one described for NadB
from E. coli [8]. After sonication in lysis buffer (50 mm
potassium phosphate buffer, pH 8.0, 20% glycerol, 1 mm
phenylmethanesulfonyl fluoride, 0.1 mg ÆmL
)1
DNase,
0.1 mgÆmL
)1
RNase, 1 mm EDTA, plus protease inhibitor
cocktail Complete Mini; Roche Diagnostics, Mannheim,
Germany), the homogenate was centrifuged at 27 000 g for

45 min at 4 °C. Solid FAD was added to the supernatant,
and the protein was precipitated by slow addition of
ammonium sulfate at 4 °C up to 25% saturation. Follow-
ing centrifugation (27 000 g for 45 min at 4 °C), the
precipitate was solubilized in 50 mm potassium phos-
phate buffer (pH 8.0) containing 20% glycerol, dialyzed
overnight against the same buffer containing 1 mm
I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5103
phenylmethanesulfonyl fluoride, and loaded on a DEAE
Sepharose column (12 · 1.5 cm) (GE Healthcare) equili-
brated in the same buffer. Pure NadB was eluted with a
linear gradient from 50 mm to 300 mm potassium
phosphate buffer (pH 8.0), 20% glycerol, and 1 mm
phenylmethanesulfonyl fluoride.
NadB apoprotein preparation
Apoprotein was prepared by incubating the holoenzyme
with charcoal and 3 m KBr in 50 mm potassium phosphate
buffer (pH 8.0) and 20% glycerol at 4 ° C for 1 h. The
apoform was collected by centrifugation at 10 000 g for
5 min at 4 ° C as in Mortarino et al. [8]. KBr was removed
using a PD10 column equilibrated in 50 mm potassium
phosphate buffer (pH 8.0) and 20% glycerol. The dissocia-
tion constant for FAD was determined as in Mortarino
et al. [8].
Binding to NadB of dicarboxylic compounds and
of the product iminoaspartate
The dissociation constants for dicarboxylic ligands were
measured spectrophotometrically by addition of small
volumes of concentrated stock solutions to samples con-

taining about 10–25 lm holoenzyme at 25 °C. The K
d
for
the product iminoaspartate was determined as described in
Mortarino et al. [8]. Iminoaspartate was produced by an
enzymatic system consisting of d-aspartate ⁄ d-aspartate oxi-
dase to produce iminoaspartate in situ free of excess
reagents, using a concentration of d-aspartate (300 lm) that
was low enough to avoid the formation of the complex
with NadB. The amount of NadB–iminoaspartate complex
can be directly estimated from the increase in absorbance
at 495 nm. The k
off
of iminoaspartate from NadB was mea-
sured as described in Mortarino et al. [8]. In detail, NadB
from B. subtilis was incubated in 50 mm potassium phos-
phate buffer (pH 8) and 20% glycerol containing 10 mm
oxaloacetate and 0.3 m ammonium sulfate to produce
iminoaspartate. Following removal of excess reagents by
gel filtration on a PD10 column, the release of iminoaspar-
tate from the complex with NadB was followed at 495 nm
both in the presence and in the absence of a stoichiometric
amount of NadA.
NadB activity assays
Rates of the enzyme-catalyzed oxidation of l-aspartate by
0.24 mm oxygen at 25 °C were measured in a coupled assay
by following the oxidation of o-dianisidine at 436 nm in
50 mm Hepes buffer (pH 8.0). The reaction mixture con-
tained 20 lm FAD, 10 lgÆmL
)1

o-dianisidine, 64 lgÆmL
)1
horseradish peroxidase, and variable amounts of the
enzyme and l-aspartate [9]. Succinate oxidase activity was
checked by using 10 mm succinate instead of l-aspartate in
the assay mixture.
Fumarate reductase activity was measured in anaerobic
conditions by following at 550 nm the reoxidation of benzyl
viologen previously reduced by sodium dithionite as previ-
ously described [14]. This assay was carried out in 50 mm
potassium phosphate buffer (pH 8.0), 20% glycerol, and
0.1 mm EDTA. The extinction coefficient used for the
reduced benzyl viologen at 550 nm was 7800 m
)1
Æcm
)1
.
l-Aspartate–fumarate oxidoreductase activity was
recorded by measuring the formation of oxaloacetate
derived from spontaneous hydrolysis of the iminoaspartate
produced by the reaction, as described in Yamada et al.
[41]. The complete reaction mixture contained 50 mm
potassium phosphate buffer (pH 8.0), 20% glycerol, 10 lm
FAD, 3 lg of NadB, and various amounts of l-aspartate
and fumarate, in a final volume of 0.5 mL. The mixture
was incubated at 37 °C in an anaerobic cuvette with shak-
ing for 30 min. After addition of 0.2 mL of 25% trichloro-
acetic acid and centrifugation at 10 000 g for 5 min,
0.5 mL of the supernatant was mixed with 0.1 mL of 0.1%
2,4-dinitrophenylhydrazine in 2 m HCl and incubated for

10 min at 37 °C; then, 0.4 mL of 3.75 m NaOH was added
to the solution, which, after being left to stand for 10 min,
was centrifuged at 10 000 g for 5 min. The absorbance of
the supernatant was measured at 445 nm, and the activity
was calculated using an extinction coefficient of
3846 m
)1
Æcm
)1
.
Gel filtration
Gel filtration was performed at room temperature with a
Superose 12 HR 10 ⁄ 30 FPLC column (GE Healthcare), at
a flow rate of 0.7 mLÆmin
)1
,in50mm potassium phosphate
buffer (pH 8.0), in the absence and in the presence of up to
0.45 m NaCl, under both aerobic and anaerobic conditions.
In the latter case, the buffer was made anaerobic by
bubbling the buffer with N
2
for more than 30 min and
then adding 1 mm sodium dithionite [8]. To analyze the
NadA–NadB complex, the two proteins were mixed with
stoichiometries of 1 : 1 or 2 : 1 NadA ⁄ NadB, and were
incubated for 30 min at room temperature under aerobic
conditions or under anaerobic conditions in a glove box
before gel filtration. Samples eluted from the column were
analyzed by SDS ⁄ PAGE.
Affinity chromatography

NadA–His immobilized on an Ni
2+
–nitrilotriacetic acid
column (Qiagen, Milano, Italy) was prepared as reported
above. Different amounts of purified B. subtilis NadB
(94 lg and 157 lg) were incubated with the resin under
anaerobic or aerobic conditions at room temperature for
30 min in 50 mm Tris ⁄ HCl (pH 8.0) and 10 mm b-mercap-
NadA and NadB from B. subtilis I. Marinoni et al.
5104 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
toethanol. The columns was washed four times with 50 mm
Tris ⁄ HCl, 300 mm NaCl, 30 mm imidazole (pH 8.0), 1 mm
phenylmethanesulfonyl fluoride, 10 mm b -mercaptoethanol,
and 20% glycerol, and eluted in 50 mm Tris ⁄ HCl, 300 mm
NaCl, 250 mm imidazole (pH 8.0), 1 mm phenyl-
methanesulfonyl fluoride, 10 mm b-mercaptoethanol, and
20% glycerol. The eluted fractions were subjected to
SDS ⁄ PAGE and electroblotted in order to verify the iden-
tity of the eluted protein by N-terminal sequence analysis.
To exclude the possibility that NadB binds the resin by
itself, NadB alone was loaded on the Ni
2+
–nitrilotriacetic
acid columns. The same experiments were also repeated
using GST–NadA instead of the protein carrying the His-
tag at the C-terminus. In this case, the glutathione–Sepha-
rose column was washed four times with 50 mm Tris ⁄ HCl
(pH 8.0), 300 mm NaCl, 1 mm phenylmethanesulfonyl fluo-
ride, 10 mm b-mercaptoethanol, and 20% glycerol, and
eluted by adding 10 mm glutathione to the buffer.

Miscellaneous methods
N-terminal amino acid sequences were determined using a
Procise sequencer (Applied Biosystems, Foster City, CA,
USA) following blotting on poly(vinylidene difluoride)
membranes.
The protein content was determined by the method of
Bradford or by UV-absorption spectroscopy at 280 nm
using extinction coefficients of 1.3 or 1.7 for 1 mgÆmL
)1
solutions of pure NadA or NadB, respectively. Unless
otherwise specified, all experiments were performed in
50 mm potassium phosphate buffer (pH 8.0) and 20% glyc-
erol to keep NadB in solution, as the absence of phosphate
or glycerol in the buffer resulted in considerable precipita-
tion of the protein. Absorption spectra were measured with
a Hewlett-Packard 8453 diode array spectrophotometer.
Fluorescence spectra were recorded with an FP-750 Jasco
spectrofluorimeter (JASCO Europe, Cremella, Italy). HPLC
purification of FAD for the fluorescence experiments was
performed as described in Mortarino et al. [8].
Purification of NadB from E. coli to be used in the
NadA activity assays and of recombinant bovine d-aspar-
tate oxidase overexpressed in E. coli to produce in situ
iminoaspartate were performed as previously described
(Mortarino et al. [8] and Negri et al. [42], respectively).
Acknowledgements
This work was supported by PRIN 2005, MIUR (Min-
istero per l’Universita
`
e la Ricerca Scientifica e Tecno-

logica, Italy) and FIRST 2007 (University of Milano,
Italy), FAR 2005 and FAR 2006 (University of Pavia,
Italy). We thank E. Andreoli for expert and careful
technical support, N. Marchesi and F. Rocchi for help
in the construction of some of the B. subtilis nadA
mutants, and F. Corniola for assistance in figure prep-
aration.
References
1 Berger F, Ramirez-Hernandez MH & Ziegler M (2004)
The new life of a centenarian: signaling functions of
NAD(P). Trends Biochem Sci 29, 111–118.
2 Nasu S, Wicks FD & Gholson RK (1982) l-aspartate
oxidase, a newly discovered enzyme of Escherichia coli,
is the B protein of quinolinate synthetase. J Biol Chem
257, 626–632.
3 Gerdes SY, Scholle MD, D’Souza M, Bernal A, Baev
MV, Farrell M, Kurnasov OV, Daugherty MD, Mseeh
F, Polanuyer BM et al. (2002) From genetic footprint-
ing to antimicrobial drug targets: examples in cofactor
biosynthetic pathways. J Bacteriol 184, 4555–4572.
4 Prunier AL, Schuch R, Fernandez RE, Mumy KL,
Kohler H, McCormick BA & Maurelli AT (2007) nadA
and nadB of Shigella flexneri 5a are antivirulence loci
responsible for the synthesis of quinolinate, a small
molecule inhibitor of Shigella pathogenicity. Microbio-
logy 153, 2363–2372.
5 Katoh A, Uenohara K, Akita M & Hashimoto T
(2006) Early steps in the biosynthesis of NAD in
Arabidopsis start with aspartate and occur in the
plastid. Plant Physiol 141, 851–857.

6 Noctor G, Queval G & Gakie
`
re B (2006) NAD(P)
synthesis and pyridine nucleotide cycling in plants and
their potential importance in stress conditions. J Exp
Bot 57, 1603–1620.
7 Murthy NM, Ollagnier-de-Choudens S, Sanakis Y,
Abdel-Ghany SE, Rousset C, Ye H, Fontecave M,
Pilon-Smits EA & Pilon M (2007) Characterization of
Arabidopsis thaliana SufE2 and SufE3: functions in
chloroplast iron–sulfur cluster assembly and NAD
synthesis. J Biol Chem 282, 18254–18264.
8 Mortarino M, Negri A, Tedeschi G, Simonic T, Duga
S, Gassen GH & Ronchi S (1996) l-aspartate oxidase
from Escherichia coli. I. Characterization of coenzyme
binding and product inhibition. Eur J Biochem 239,
418–426.
9 Tedeschi G, Negri A, Mortarino M, Ceciliani F, Simon-
ic T, Faotto L & Ronchi S (1996) l-aspartate oxidase
from Escherichia coli. II. Interaction with C4-dicarboxy-
lic acids and identification of a novel l-aspartate:fuma-
rate oxidoreductase activity. Eur J Biochem 239 ,
427–433.
10 Tedeschi G, Zetta L, Negri A, Mortarino M, Ceciliani
F & Ronchi S (1997) Redox potentials and quinone
reductase activity of l-aspartate oxidase from E. coli.
Biochemistry 36, 16221–16230.
11 Bacchella L, Lina C, Todone F, Negri A, Tedeschi G,
Ronchi S & Mattevi A (1999) Crystallization of
I. Marinoni et al. NadA and NadB from B. subtilis

FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5105
l-aspartate oxidase, the first enzyme in the bacterial
de novo biosynthesis of NAD. Acta Crystallogr 55,
549–551.
12 Tedeschi G, Negri A, Ceciliani F, Mattevi A & Ronchi
S (1999) Structural characterization of l-aspartate
oxidase and identification of an interdomain loop by
limited proteolysis. Eur J Biochem 260, 896–903.
13 Mattevi A, Tedeschi G, Bacchella L, Coda A, Negri A
& Ronchi S (1999) Structure of l-aspartate oxidase:
implications for the succinate dehydrogenase ⁄ fumarate
reductase oxidoreductase family. Structure 7, 745–756.
14 Tedeschi G, Ronchi S, Simonic T, Treu C, Mattevi A &
Negri A (2001) Probing the active site of l-aspartate
oxidase by site-directed mutagenesis: role of basic resi-
dues in fumarate reduction. Biochemistry 40, 4738–4744.
15 Bossi RT, Negri A, Tedeschi G & Mattevi A (2002)
Structure of FAD-bound l-aspartate oxidase: insight
into substrate specificity and catalysis. Biochemistry 41,
3018–3024.
16 Sakuraba H, Satomura T, Kawakami R, Yamamoto S,
Kawarabayasi Y, Kikuchi H & Ohshima T (2002)
l-aspartate oxidase is present in the anaerobic hyper-
thermophilic archaeon Pyrococcus horikoshii OT-3:
characteristics and role in the de novo biosynthesis of
nicotinamide adenine dinucleotide proposed by genome
sequencing. Extremophiles 6, 275–281.
17 Sakubara H, Yoneda K, Asai I, Tsuge H, Katunuma N
& Ohshima T (2008) Structure of l-aspartate oxidase
from the hyperthermophilic archaeon Sulfolobus toko-

daii. Biochim Biophys Acta 1784, 563–571.
18 Yang Z, Savchenko A, Yakunin A, Zhang R, Edwards
A, Arrowsmith C & Tong L (2003) Aspartate dehydro-
genase, a novel enzyme identified from structural and
functional studies of TM1643. J Biol Chem 278, 8804–
8808.
19 Ceciliani F, Caramori T, Ronchi S, Tedeschi G, Morta-
rino M & Galizzi G (2000) Cloning, overexpression and
purification of E. coli quinolinate synthetase. Protein
Expr Purif 18, 64–70.
20 Cicchillo RM, Tu L, Stromberg JA, Hoffart LM, Krebs
C & Booker SJ (2005) Escherichia coli quinolinate
synthetase does indeed harbor a [4Fe–4S] cluster. JAm
Chem Soc 127, 7310–7311.
21 Ollagnier-de Choudens S, Loiseau L, Sanakis Y, Barras
F & Fontecave M (2005) Quinolinate synthetase, an
iron–sulfur enzyme in NAD biosynthesis. FEBS Lett
579, 3737–3743.
22 Sakuraba H, Tsuge H, Yoneda K, Katunuma N &
Ohshima T (2005) Crystal structure of the NAD biosyn-
thetic enzyme quinolinate synthase. J Biol Chem 280
,
26645–26648.
23 Gardner PR & Fridovich I (1991) Quinolinate synthe-
tase: the oxygen-sensitive site of de novo NAD(P)+
biosynthesis. Arch Biochem Biophys 284, 106–111.
24 Sun D & Setlow P (1993) Cloning, nucleotide sequence,
and regulation of the Bacillus subtilis nadB gene and a
nifS-like gene, both of which are essential for NAD
biosynthesis. J Bacteriol 175, 1423–1432.

25 Seemann M, Wegner P, Schu
¨
nemann V, Tse Sum Bui
B, Wolff M, Marquet A, Trautwein AX & Rohmer M
(2005) Isoprenoid biosynthesis in chloroplasts via the
methylerythritol phosphate pathway: the (E)-4-hydroxy-
3-methylbut-2-enyl diphosphate synthase (GcpE) from
Arabidopsis thaliana is a [4Fe–4S] protein. J Biol Inorg
Chem 10, 131–137.
26 Layer G, Teschner T, Schu
¨
nemann V, Breckau D,
Heathrote P, Trautwein AX & Jahn D (2005) Radical
S-adenosylmethionine enzyme coproporphyrinogen III
oxidase HemN: functional features of the [4Fe–4S] clus-
ter and the two bound S-adenosyl-l-methionines. J Biol
Chem 280, 29038–29046.
27 Tse Sum Bui B, Benda R, Schu
¨
nemann V, Florentin D,
Trautwein AX & Marquet A (2003) Fate of the (2Fe–
2S)(2 + ) cluster of Escherichia coli biotin synthase
during reaction: a Mo
¨
ssbauer characterization.
Biochemistry 42, 8791–8798.
28 Beinert H, Kennedy MC & Stout CD (1996) Aconitase
as iron–sulfur protein, enzyme, and iron-regulatory
protein. Chem Rev 96, 2335–2373.
29 Wasby CJ, Hong W, Broderick WE, Cheek J, Ortillo

D, Bruderick JB & Hoffmann BM (2002) Catalytically
active [4Fe–4S]
+
cluster of pyruvate formate-lyase acti-
vating enzyme. J Am Chem Soc 124 , 3143–3151.
30 Ollagnier S, Meier C, Mulliez E, Gaillard J, Schu
¨
ne-
mann V, Trautwein AX, Mattioli T, Lutz M & Fonte-
cave M (1999) Assembly of 2Fe–2S and [4Fe–4S]
clusters in the anaerobic ribonucleotide reductase from
Escherichia coli . J Am Chem Soc 121, 6344–6350.
31 Bates DM, Popescu CV, Khoroshilova N, Vogt K,
Beinert H, Mu
¨
nck E & Kiley PJ (2000) Substitution of
leucine 28 with histidine in the Escherichia coli tran-
scription factor FNR results in increased stability of the
[4Fe–4S](2 + ) cluster to oxygen. J Biol Chem 275,
6234–6240.
32 Gattilker A, Michoud K, Rivoire C, Auchincloss AH,
Coudert E, Lima T, Kersey P, Pagni M, Sigrist CJA,
Lachaizer C et al. (2002) Automated annotation of
microbial proteomes in SWISS-PROT. Comput Biol
Chem 27, 49–58.
33 Arnaud M, Chastanet A & De
´
barbouille
´
M (2004) New

vector for efficient allelic replacement in naturally non-
transformable, low-GC content Gram-positive bacteria.
Appl Environ Microbiol 70, 6887–6891.
34 Griffith GR, Chandler JLR & Gholson RK (1975)
Studies on the de novo biosynthesis of NAD in Escheri-
chia coli. The separation of the nadB gene product from
the nadA gene product and its purification. Eur J
Biochem 54, 239–245.
NadA and NadB from B. subtilis I. Marinoni et al.
5106 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS
35 Draczynska-Lusiak B & Brown OR (1992) Protein A of
quinolinate synthetase is the site of oxygen poisoning of
pyridine nucleotide coenzyme synthesis in Escherichia
coli. Free Radic Biol Med 13, 689–693.
36 Mattevi A (2006) A close look at NAD biosynthesis.
Nat Struct Mol Biol 13, 563–564.
37 Flint DH, Emptage MH, Finnegan MG, Fu W &
Johnson MK (1993) The role and properties of the
iron–sulfur cluster in Escherichia coli dihydroxy-acid
dehydratase. J Biol Chem 268, 14732–14742.
38 Gu
¨
tlich P, Link R & Trautwein A (1978) Mo
¨
ssbauer
Spectroscopy and Transition Metal Chemistry (Inor-
ganic Chemistry Concepts, Vol. 3). Springer, New
York.
39 Vanoni MA, Edmonson DE, Zanetti G & Curti B
(1992) Characterization of the flavins and the iron–

sulfur centers of glutamate synthase from Azospirillum
brasilense by absorption, circular dichroism, and elec-
tron paramagnetic resonance spectroscopies. Biochemis-
try 31, 613–4623.
40 Rabinowitz JC (1978) Analysis of acid-labile sulfide and
sulfhydryl groups. Methods Enzymol 53, 275–277.
41 Yamada R, Nagasaki H, Wabayashi Y & Iwashima A
(1988) Presence of d-aspartate oxidase in rat liver and
mouse tissues. Biochim Biophys Acta 965, 202–205.
42 Negri A, Tedeschi G, Ceciliani F & Ronchi S (1999)
Purification of beef kidney d-aspartate oxidase overex-
pressed in E. coli and characterizaztion of its redox
potentials and oxidative activity towards agonists of
excitatory amino acid receptors. Biochim Biophys Acta
1431, 212–222.
43 Davis BD & Mingioli ES (1953) Aromatic biosynthesis.
VII. Accumulation of two derivatives of shikimic acid
by bacterial mutants. J Bacteriol 66, 129–136.
Supporting information
The following supplementary material is available:
Table S1. Primers used for cloning, in the expression
or allelic switch vectors, and for site-directed muta-
genesis.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
material supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.

I. Marinoni et al. NadA and NadB from B. subtilis
FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5107