Báo cáo khoa học: Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases potx

Enzymatic oxidation of NADP
+
to its 4-oxo derivative is a
side-reaction displayed only by the adrenodoxin reductase
type of ferredoxin-NADP
+
reductases
Matteo de Rosa
1
, Andrea Pennati
1
, Vittorio Pandini
1
, Enrico Monzani
2
, Giuliana Zanetti
1
and Alessandro Aliverti
1
1 Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita
`
degli Studi di Milano, Italy
2 Dipartimento di Chimica Generale, Universita
`
degli Studi di Pavia, Italy
Ferredoxin-NADP
+
reductases (FNRs, EC 1.18.1.2)
can be classified into two phylogenetically distinct
subgroups: the mitochondrial-type and the plastid-type
(or plant-type) enzymes [1]. The prototype of the

former enzymes is the mammalian adrenodoxin reduc-
tase (AdR), whereas the latter subgroup is best
exemplified by the photosynthetic FNR. Although both
FNR types catalyze the same physiologic reaction, i.e.
Keywords
adrenodoxin reductase; 3-carboxamide-4-
pyridone adenine dinucleotide phosphate;
flavoprotein; Mycobacterium tuberculosis;
NADP derivative
Correspondence
A. Aliverti, Dipartimento di Scienze
Biomolecolari e Biotecnologie, via Celoria 26,
20133 Milano, Italy
Fax: +39 02 50314895
Tel: +39 02 50314897
E-mail:
Website: />(Received 19 March 2007, revised 7 May
2007, accepted 11 June 2007)
doi:10.1111/j.1742-4658.2007.05934.x
We have previously shown that Mycobacterium tuberculosis FprA, an
NADPH-ferredoxin reductase homologous to mammalian adrenodoxin
reductase, promotes the oxidation of NADP
+
to its 4-oxo derivative 3-car-
boxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti
A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS,
Heck AJ, Rizzi M et al. (2002) Biochemistry 41, 8807–8818]. Here, we pro-
vide a detailed study of this unusual enzyme reaction, showing that it
occurs at a very slow rate (0.14 h
)1

), requires the participation of the
enzyme-bound FAD, and is regiospecific in affecting only the C4 of the
NADP nicotinamide ring. By protein engineering, we excluded the involve-
ment in catalysis of residues Glu214 and His57, previously suggested to be
implicated on the basis of their localization in the three-dimensional struc-
ture of the enzyme. Our results substantiate a catalytic mechanism for
3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in
which the initial and rate-determining step is the nucleophilic attack of the
nicotinamide moiety by an active site water molecule. Whereas plant-type
ferredoxin reductases were unable to oxidize NADP
+
, the mammalian
adrenodoxin reductase also catalyzed this unusual reaction. Thus, the
3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reac-
tion seems to be a peculiar feature of the mitochondrial type of ferredoxin
reductases, possibly reflecting conserved properties of their active sites.
Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleo-
tide phosphate is good ligand and a competitive inhibitor of various dehy-
drogenases, making this nucleotide analog a useful tool for the
characterization of the cosubstrate-binding site of NADPH-dependent
enzymes.
Abbreviations
AdR, adrenodoxin reductase; Amplex Red, 10-acetyl-3,7-dihydroxyphenoxazine; amu, atomic mass units; CT1, charge transfer complex
between FprA and NADPH; FNR, ferredoxin-NADP
+
reductase; INT, iodo-nitro-tetrazolium chloride; NADPO, 3-carboxamide-4-pyridone
adenine dinucleotide phosphate; NMNO, 3-carboxamide-4-pyridone mononucleotide; 2P-AMP, 2¢-phospho-AMP.
3998 FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS
the transfer of a couple of electrons from NADPH
to two successive ferredoxin molecules, they mark-

edly differ in their structure and functional properties
[2,3].
In the recent past, we have obtained the crystal
structure of FprA, a Mycobacterium tuberculosis
homolog of AdR (41% identity spread over the entire
polypeptide length) at a very high (1.05 A
˚
) resolution
[4]. This accomplishment, together with the fact that
the structures of AdR and FprA are very similar,
made the bacterial protein an ideal representative of
mitochondrial-type FNRs for structure–function rela-
tionship studies. The atomic resolution of the FprA
structure allowed us to discover that this enzyme, in
addition to the well-known NADPH-dependent ferre-
doxin reduction, catalyzed an unprecedented reaction,
i.e. the oxidation of NADP
+
to yield its 4-oxo deriv-
ative, which was named 3-carboxamide-4-pyridone
adenine dinucleotide phosphate (NADPO). The first
evidence that such a reaction was occurring was the
observation that crystals of FprA grown in the pres-
ence of NADP
+
contained NADPO bound to the act-
ive site instead of NADP
+
[4]. Comparison of the
FprA–NADPO and FprA–NADPH structures clearly

showed that in the latter complex an additional
ordered water molecule (water 1) was sitting in the act-
ive site in a position very close to that occupied by
the carbonyl oxygen atom of NADPO in the former
complex [4]. This observation prompted us to propose
the hypothetical mechanism for the FprA-catalyzed
NADPO formation reaction depicted in Fig. 1.
NADP
+
oxidation is initiated by water 1 addition to
the electron-poor C4 atom of the nicotinamide ring of
NADP
+
. His57 and Glu214 have been previously pro-
posed to increase the nucleophilicity of the water mole-
cule by favoring its deprotonation [4]. Both residues,
highly conserved in AdR-like enzymes, have been
recently changed to nonionizable ones by site-directed
mutagenesis [5]. Characterization of the resulting FprA
variants allowed us to conclude that His57 but not
Glu214 played a significant role in the physiologic,
NADPH-dependent activity of FprA. However, crys-
tals of FprA-H57Q grown in the presence of NADP
+
again displayed NADPO as the bound nucleotide by
X-ray diffractometry [5], showing that the H57Q muta-
tion did not abolish the NADP
+
oxidation activity of
FprA.

The previous studies summarized above only gave a
qualitative evidence of the ability of FprA to catalyze
the production of NADPO. In the present article, we
provide the first quantitative description of this reac-
tion, along with a detailed analysis of the spectroscopic
properties of the product of NADP
+
oxidation.
Results and Discussion
NADPO isolation, quantitation, and spectral
characterization
In order to study the kinetics of NADP
+
oxidation to
NADPO catalyzed by FprA, an NADPO assay
method was required. After testing different analytical
chromatographic procedures, we found the ion
exchange method described by Orr & Blanchard to be
particularly reliable and robust for our purposes [6].
Inclusion of AMP as an internal standard increased
precision and accuracy, and replacement of the ori-
ginal mobile phase with a volatile ammonium formate
buffer allowed the recovery of purified nucleotide as
salt-free preparations after vacuum drying. Figure 2
shows the typical analysis of aliquots sampled at
increasing incubation times from a reaction mixture
where FprA was incubated with NADP
+
in air. As
Fig. 1. Hypothetical mechanism of the reductive half-reaction of

the catalytic cycle of FprA in the oxidation of NADP
+
to yield
NADPO. The reaction scheme was based on the crystal structures
of the FprA–NADPO and FprA–NADPH complexes [4]. The depicted
water molecule is referred to in the text as water 1. B1 and B2 rep-
resent hypothetical groups acting as base catalysts. B1 has been
proposed to be the imidazyl of His57 [4].
M. de Rosa et al. Oxidation of NADP
+
to its 4-oxo derivative
FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS 3999
can be seen, incubation over several hours resulted in
a gradual decrease of the NADP
+
peak while a single
new peak appeared and progressively increased in
intensity. By tandem MS, the latter peak was found to
unambiguously contain an NADP derivative bearing
an oxygen atom bound to the nicotinamide ring. Previ-
ously, the same NADP
+
modification was observed by
MS analysis of the whole reaction mixture [4]. How-
ever, even though only 4-oxo-NADP was observed by
X-ray crystallography in complex with FprA, in princi-
ple it cannot be excluded that FprA could also intro-
duce an oxygen atom at other positions of the
nicotinamide ring of NADP
+

, leading to other nucleo-
tide derivatives with a lower affinity for the enzyme
active site. Thus, it was of interest to ascertain the
identity of the isolated compound by a detailed spect-
roscopic characterization. Values of 17 100 and
15 600 m
)1
Æcm
)1
were found for the extinction coeffi-
cient of the NADP derivative at 260 and 254 nm,
respectively, as calculated by determining the concen-
tration of the nucleotide on the basis of the phosphate
released by alkaline phosphatase treatment. The
absorbance spectrum of the nucleotide is shown in
Fig. 3A in comparison to those of NADP
+
and
NADPH. A peculiar feature of the modified NADP
+
is a relatively high absorption in the 280–310 nm
region. To study the effect of pH on its spectral prop-
erties, the adenylate portion of the modified nucleotide
was removed by enzymatically splitting the pyrophos-
phate link in order to get rid of its large absorbance
contribution. As shown in Fig. 3B, the spectrum of
chromatographically purified NMN derivative under-
goes a large spectral transition when the pH is
decreased from 7.7 to 1.0. The two spectra are very
similar to those described for N -methyl-4-pyridone-

5-carboxamide, and completely different from those of
N-methyl-2-pyridone-5-carboxamide, under similar pH
conditions [7]. Furthermore, the isolated NADP deriv-
ative was found to lack any fluorescence when excited
with light in the UV region. This observation is rele-
vant for excluding the presence of species modified in
position 6 of the nicotinamide ring, because, unlike the
compounds with the oxo group in positions 2 and 4,
N-methyl-6-pyridone-5-carboxamide has been shown
to emit blue light by fluorescence [8]. We exclude the
formation of the species modified in position 3 of the
nicotinamide ring because of the poor reactivity of this
position towards nucleophilic attack. Indeed, the posit-
ive charge of the pyridinium moiety favors attack by
nucleophiles at positions 2 and 4 under mild condi-
tions [9]. Furthermore, and more importantly, the
modification at position 3 can be excluded because the
resulting N-substituted 3-oxo-nicotinamide moiety
would not be neutral, resulting in a dinucleotide with
spectral and chromatographic properties expected to
be markedly different from those of 4-oxo-NADP.
These data allow us to conclude that FprA does not
Fig. 2. Anion exchange chromatographic pattern of NADP
+
oxida-
tion reaction mixtures. NADP
+
at 500 lM was reacted with O
2
at

its air equilibrium concentration (c. 250 l
M) in the presence of
100 l
M FprA at 25 °C. Forty-microliter aliquots were analyzed by
high-performance chromatography as described in Experimental
procedures. Reaction times were 0 h (dotted trace), 3 h (dashed
trace), 6 h (dot-dashed trace), and 24 h (continuous trace).
Fig. 3. Extinction coefficients and absorption spectra of NADPO
and NMNO. (A) Dinucleotides in 20 m
M Tris ⁄ HCl (pH 7.7). (B)
NMNO in 20 m
M Tris ⁄ HCl (pH 7.7), and after bringing the pH to 1
by the addition of HCl.
Oxidation of NADP
+
to its 4-oxo derivative M. de Rosa et al.
4000 FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS
produce detectable amounts of NADP derivatives
bearing a carbonylic oxygen at sites other than posi-
tion 4 of the nicotinamide ring, indicating that the
NADP
+
oxidation reaction is highly regiospecific.
Kinetics of NADPO formation as catalyzed by
FprA and AdR
Figure 4A shows the time courses of NADP
+
oxida-
tion to NADPO catalyzed by FprA or AdR in the
presence of air oxygen. Clearly, both enzymes are able

to catalyze this reaction. Studies at various NADP
+
concentrations were performed, showing that the sub-
strate concentration of 500 lm was fully saturating.
The initial rate of NADPO formation promoted by
AdR was slightly lower (75%) than that of FprA
under the same conditions. A peculiar feature of the
NADP
+
oxidation kinetics is the progressive decrease
in the reaction rate. When FprA was the catalyst, this
behavior was particularly marked: NADPO formation
sharply decreased after the first enzyme turnover. We
excluded the possibility that this was a consequence of
enzyme denaturation by assaying the enzyme during
incubation. A reasonable explanation is that NADPO
was acting as a competitive inhibitor of the enzyme
with respect to NADP
+
(see below). In the case of
FprA, the reaction was studied at different enzyme
concentrations. As shown in Fig. 4B, the initial rate of
the reaction was proportional to FprA concentration,
showing that the NADP
+
oxidation, although very
slow, was strictly enzyme-dependent. The NADP
+
oxidation reaction catalyzed by FprA was much slower
(0.14 h

)1
) than that of its NADPH-dependent,
physiologic reaction (336 min
)1
when Mycobacterium
smegmatis FdxA was used as electron acceptor [10]).
Plant-type FNRs do not catalyze NADPO
formation
To verify whether NADP
+
oxidation to yield NADPO
was a common feature of the members of the FNR
class of enzymes, we assayed Toxoplasma gondii FNR
[11], Plasmodium falciparum FNR [12] and spinach
(Spinacia oleracea) leaf FNR [13] (which all are plant-
type FNRs) for their ability to catalyze this reaction,
and found them to be completely inactive. Although
FNRs from other sources should be assayed before
drawing general conclusions, it is suggested that
NADP
+
oxidation to NADPO most probably repre-
sents a unique feature of AdR-type FNRs and reflects
very specific organization and reactivity of their active
sites.
Role of O
2
in enzyme-catalyzed NADP
+
oxidation

When the O
2
-dependent NADPO production reaction
catalyzed by FprA was allowed to proceed in the pres-
ence of excess peroxidase and its fluorogenic substrate
10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), a
progressive increase in fluorescence emission at 585 nm
was observed (not shown), indicating accumulation of
the fluorescent product resorufin. No fluorescence built
up in the absence of peroxidase, allowing us to attrib-
ute resorufin formation entirely to the 1 : 1 reaction
between H
2
O
2
and Amplex Red catalyzed by the per-
oxidase. This conclusion was confirmed by the effect
of the presence of catalase in the reaction mixture,
which completely abolished resorufin formation. On
the basis of the quantum yield experimentally deter-
mined for resorufin, a rate of 0.08 mol H
2
O
2
(mol
FprA)
)1
was calculated, a value comparable to the
rate of NADPO formation. In addition to H
2

O
2
,a
small amount of superoxide was apparently produced
in the reaction, as judged from the slight increase in
the rate of fluorescence appearance after superoxide
dismutase addition. However, it could not be excluded
that the peroxide ⁄ superoxide ratio was substantially
lower than that found, as the low rate of the reaction
would allow enough time for spontaneous disproportio-
nation of O
2
–
to H
2
O
2
and O
2
. In any case, the produc-
tion of H
2
O
2
and ⁄ or O
2
–
strongly supports the FAD
prosthetic group as the direct oxidant of the nicotina-
mide ring, as these species are the usual products of

FADH
2
reoxidation by O
2
in most flavoproteins.
To verify whether the direct reaction between
NADP
+
and O
2
was not required in the enzymatic
production of NADPO, the latter reaction was studied
in the presence of electron acceptors different from O
2
.
NADP
+
was incubated with FprA under anaerobic
Fig. 4. (A) Time-courses of NADPO production catalyzed by FprA
and AdR in the presence of O
2
. The reaction was carried out at
25 °C in the presence of 100 l
M FprA (filled circles) or bovine AdR
(open circles) and 500 l
M NADP
+
. A control experiment was per-
formed, omitting the enzyme (squares). (B) Initial rate of NADPO
production calculated over the first 6 h of the reaction plotted as a

function of the enzyme concentration.
M. de Rosa et al. Oxidation of NADP
+
to its 4-oxo derivative
FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS 4001
conditions in the presence or in the absence of
K
3
Fe(CN)
6
, an artificial electron acceptor of the
enzyme. As shown in Fig. 5A, as NADP
+
was added
to the reaction mixture, a progressive bleaching of the
absorbance contributed by ferricyanide was observed,
indicating its reduction to ferrocyanide. The spectrum
of enzyme-bound FAD remained unaltered, until all
the ferricyanide was consumed. Starting from this
point, the FAD spectrum undergoes a progressive per-
turbation (not shown), similar to that observed by
incubating FprA with NADP
+
in the absence of ferri-
cyanide (Fig. 5B). The ability of FprA to carry on
NADP
+
oxidation using either O
2
or ferricyanide sup-

ports our previous proposal, made on the basis of
structural data [4], that the mechanism of NADPO
formation can be split in two half-reactions: the first
leads to NADPO formation coupled to FAD reduc-
tion; and the second consists of the reoxidation of
FADH
2
by O
2
or other oxidants (Fig. 1). With the
aim of trapping possible intermediates produced in the
first half-reaction, the reaction between NADP
+
and
FprA was studied in the absence of any oxidant, thus
preventing the second, oxidative, half-reaction of the
catalytic cycle. During incubation, the A
340
of the mix-
ture progressively increased, and the visible absorbance
spectrum of the enzyme-bound FAD underwent a pro-
gressive partial bleaching, leading to a final stable
spectrum strongly reminiscent of that of the charge
transfer complex between NADPH and oxidized FprA
[10]. The time course of the FAD spectral change
approximately followed a single exponential decay
equation (inset of Fig. 5B) with a first-order rate con-
stant of 0.095 ± 0.009 h
)1
, a value similar to the rate

of NADPO formation measured under aerobic condi-
tions. The enzyme present in the endpoint reaction
mixture was denatured and precipitated before admit-
ting air into the anaerobic cuvette, and the enzyme-free
solution was analyzed by anion exchange chromato-
graphy as previously described. The sample was found
to contain both NADPO and NADPH. Thus, in the
absence of oxidants, FprA catalyzes the disproportio-
nation of NADP
+
according to the following equa-
tion:
2NADP
þ
þ OH
À
! NADPO þ NADPH þ H
þ
ð1Þ
As FprA has a single binding site for NADPH [4,5],
the reaction must proceed through a ping-pong mech-
anism, with the transferred electron couple being tran-
siently stored on the enzyme prosthetic group. Thus,
experimental evidence points to the involvement of
FAD in the mechanism of NADPO formation. The
observed spectral changes can be interpreted as result-
ing from the following reaction mechanism, where
E(FAD) and E(FADH
–
) represent the oxidized and

fully reduced form of FprA, respectively:
E(FAD) þ NADP
þ
! E(FAD)ÀNADP
þ
ð2Þ
E(FAD)ÀNADP
þ
þ OH
À
! E(FADH
À
ÞÀNADPO þ H
þ
ð3Þ
NADP
þ
þ E(FADH
À
ÞÀNADPO ! NADPO þ
E(FAD)ÀNADPH (CT1) ð4Þ
In the reaction shown in Eqn (4), the FprA–
NADPH complex represents a charge transfer species
(CT1) [10]. The reactions shown in Eqn (2) and Eqn
(4) are expected to be much faster than that shown in
Eqn (3), as dinucleotide binding and release to and
from FprA have never been found to be limiting steps
in reactions of the enzyme with NADPH or NADH,
which occurred at rates much higher than that of
NADPO formation [5]. Thus, the only process access-

ible to experimental observation was the conversion of
the E(FAD)–NADP
+
complex to CT1, precluding any
further dissection of the reaction mechanism by time-
Fig. 5. Spectral changes resulting from the incubation of FprA with
NADP
+
in the presence and absence of potassium ferricyanide
under anaerobic conditions. (A) FprA at c.20l
M was incubated
with 200 l
M NADP
+
and 140 lM K
3
Fe(CN)
6
at 25 °Cin20mM
Hepes ⁄ NaOH (pH 7.0), containing 100 mM NaCl and 10% glycerol,
within a gas-tight cuvette made anaerobic by several vacuum–N
2
flushing cycles. Reaction times were 0 min to 330 min. (B) Same
conditions as in (A), with the omission of K
3
Fe(CN)
6
. Reaction
times were 0 h to 19 h. Inset: absorbance at 580 nm as a function
of incubation time. The curve represents the best fit according to a

single exponential decay equation (k ¼ 0.095 h
)1
).
Oxidation of NADP
+
to its 4-oxo derivative M. de Rosa et al.
4002 FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS
resolved spectral analysis. The fact that CT1 formation
follows single-phase kinetics without the formation
of any observable transient intermediate suggests
that water molecule addition to the nicotinamide
moiety (Fig. 1) might be the limiting step of the whole
reaction.
Investigating the role of Glu214 and His57 of
FprA in the catalysis of NADPO production
As it was not possible to obtain detailed information
on the catalytic mechanism of the NADP
+
oxidation
reaction by kinetic studies, we attempted to gain
insights into the role of specific residues of FprA by
site-directed mutagenesis. On the basis of structural
data, Glu214 and His57 of FprA have been proposed
to be involved in promoting nucleophilic attack by a
water molecule on the C4 of the nicotinamide moiety
of NADP
+
[4]. The production and purification of
the enzyme variants, where these residues were
replaced with Ala or Gln, have been described else-

where [5]. The physiologic NADPH-dependent activity
of the mutant FprA forms was characterized in detail.
Unlike Glu214, which had essentially no effect on this
activity, His57 turned out to decrease by 4–5-fold the
hydride transfer rate from NADPH or NADH to the
enzyme-bound FAD [5]. Here, we have assayed FprA-
E214A and FprA-H57Q for their ability to catalyze
NADP
+
oxidation to NADPO. As shown in Fig. 6,
both mutant forms supported the production of
NADPO, with time-courses similar to that of the
wild-type enzyme. It is noteworthy that both single
mutations slightly increased the initial rate of
NADPO synthesis. At 500 lm NADP
+
, the reaction
rates were 0.21 h
)1
and 0.19 h
)1
for FprA-E214A
and FprA-H57Q, respectively. If the rate-determining
step of NADPO production is water addition, the
proposed activating role of Glu214 and His57 should
be dismissed. The crystal structure of the complex
between FprA-H57Q and NADPO has been obtained
at 1.8 A
˚
resolution [5]. Slight alterations in the posi-

tioning of the nicotinamide ring in the active site have
been observed by comparing the mutant with the
wild-type enzyme. The described 0.7 A
˚
shift of the
nicotinamide moiety could represent the structural
basis of the observed small increase in NADP
+
oxidation rate observed with this mutant.
NADPO as an inhibitor of FNRs
To the best of our knowledge, this is the first time that
enzymatic oxidation of NADP
+
has been reported
and the spectral properties of the resulting NADP
derivative have been described. It was thus of interest
to provide a first characterization of NADPO as a
possible probe for studying the NADPH-binding site
of dehydrogenases. By inhibition studies under steady-
state conditions, we have found that NADPO acts as a
competitive inhibitor with respect to NADPH on var-
ious FNRs (both photosynthetic and nonphotosynthet-
ic), with K
i
values ranging between 1 and 30 lm
(Table 1). FprA is the enzyme most strongly inhibited
by this nucleotide. To better characterize the interac-
tion of NADPO with FprA, enzyme–ligand binding
was studied by difference spectrophotometry using var-
ious nucleotides: NADPO, NADP

+
, thio-NADP
+
and 2¢-phospho-AMP (2P-AMP). The K
d
determined
for the complex between the enzyme and NADPO was
slightly but significantly lower than that measured for
NADP
+
(Table 2). As 2P-AMP, which lacks the
NMN portion of NADP
+
but still strongly binds to
FprA, yielded very weak perturbations of the absorp-
tion spectrum of the enzyme, it is clear that the intense
difference spectra of the FprA–dinucleotide complexes
are due to the alteration of the isoalloxazine microen-
vironment induced by the binding of the NMN group.
The small structural differences in the nicotinamide
rings of NADP
+
, NADPO and thio-NADP
+
resulted
in small but significant differences in the corresponding
difference spectra (Table 2). In plant FNRs, we found
a correlation between the intensity of the difference
spectrum induced by binding and the degree of
Fig. 6. Time-courses of NADPO production catalyzed by wild-type

and mutant forms of FprA. The reaction was carried out at 25 °Cin
the presence of 100 l
M wild-type FprA (filled circles), FprA-E214A
(open circles) or FprA-H57Q (squares) and 500 l
M NADP
+
in air-
equilibrated buffer.
M. de Rosa et al. Oxidation of NADP
+
to its 4-oxo derivative
FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS 4003
occupancy of the nicotinamide ring of the bound
ligand in the active site [14]. In this view, it is signifi-
cant that the perturbations induced by NADPO bind-
ing to FprA were more intense than those induced by
the other dinucleotides tested. This suggests that the
4-pyridone-5-carboximide ring of NADPO is partic-
ularly well fitted to stack over the isoalloxazine ring,
resulting in a higher occupancy than with dinucleotides
carrying other pyridine derivatives. These observations
support the hypothesis that accumulation of NADPO
would substantially inhibit its own production by
FprA, due to competition with NADP
+
for binding to
the enzyme active site.
Conclusions
In this article, we provide clear evidence that
M. tuberculosis FprA and bovine AdR, but not plant-

like FNRs, catalyze the FAD-dependent oxidation of
NADP
+
to NADPO. This enzyme activity, possibly
shared by all AdR-like enzymes, is highly regiospecif-
ic, in that it targets only the 4-position of the pyrid-
ine ring of the substrate. The very low reaction rate
tends to exclude a physiologic role for NADPO,
although at least one example exists of an NADP
derivative, i.e. nicotinic acid adenine dinucleotide
phosphate, with documented signaling functions [15].
Rather, we feel that the NADP
+
oxidation activity
of AdR-like enzymes reflects a specific and conserved
reactivity of their active sites, where a water molecule
exerts considerable strain and possibly a polarizing
effect on the C4 atom of the nicotinamide moiety of
the bound substrate. The strict interaction between a
zinc-bound water molecule and the nicotinamide ring
has been suggested to have a role in activating
NADH for efficient hydride transfer in the catalytic
cycle of horse liver alcohol dehydrogenase [16]. It can
be speculated that water 1 plays a similar role in the
active site of AdR-type FNRs, i.e. it favors
the hydride transfer between NADPH and FAD in
the physiologic reaction catalyzed by these enzymes.
When NADP
+
is the enzyme ligand, the electrophilicity

of the nicotinamide C4 promotes water 1 addition to
the nicotinamide and subsequent FAD-dependent oxi-
dation to give NADPO. It is interesting to note that
no ordered water molecules are present in the prox-
imity of the nicotinamide ring in the crystal structure
of the complexes between plant FNR and NADP
+
or NADPH [14]. This observation could provide a
rationale for the lack of NADP
+
oxidation activity
in plant-type FNRs. Further work will be required to
fully elucidate the actual role of active site water mole-
cules in modulating the reactivity of NADP
+
and
NADPH bound to FprA.
Experimental procedures
Enzymes and chemicals
Calf intestine alkaline phosphatase was obtained from GE
Healthcare (Milano, Italy). Crotalus durissus phosphodiest-
erase, beef liver catalase, superoxide dismutase from bovine
Table 1. Inhibition constants of NADPO for different FNRs. 2,6-Dichloroindophenol reductase activity was measured using either 62 nM FprA
or 2.5 n
M T. gondii FNR in 0.2 M Tris ⁄ HCl (pH 8.2) at 25 °C at a fixed concentration of 66 lM 2,6-dichloroindophenol; the NADPH concentra-
tion was varied between 0.1 and 10 l
M in the case of FprA, and between 2 and 27 lM in the case of T. gondii FNR. The concentration of
NADPO was varied between 0 and 20 l
M. INT reductase activity was measured using 2.5 nM spinach leaf FNR in 0.2 M Tris ⁄ HCl (pH 9.0),
70 m

M NaCl and 0.1% Triton X-100 at 25 °C at a fixed concentration of 100 lM INT; the NADPH concentration was varied between 2 and
27 l
M; the NADPO concentration was varied between 0 and 20 lM. All assay mixtures included an NADPH-regenerating system comprising
glucose 6-phosphate and glucose-6-phosphate dehydrogenase.
Enzyme Diaphorase reaction
K
NADPH
m
(lM)
a
k
cat
(s
)1
)
a
K
NADPO
i
(lM)
FprA NADPH fi 2,6-dichloroindophenol 0.2 ± 0.01 4.1 ± 0.05 1.2 ± 0.2
Spinach leaf FNR NADPH fi INT 5.0 ± 0.4 54 ± 1 25 ± 5
T. gondii FNR NADPH fi 2,6-dichloroindophenol 3.5 ± 0.2 64 ± 1.5 30 ± 5
a
Values reported in the table should be considered as ‘apparent’ kinetic parameters, as they were determined at a nonsaturating fixed con-
centration of the electron acceptor.
Table 2. Affinities of various nucleotides for FprA and extent of the
spectral perturbations induced by their binding to the enzyme.
Ligand K
d

(lM) De
a
(mM
)1
Æcm
)1
)
2P-AMP 3.6 ± 2 0.22 (489 nm)
NADP
+
6.3 ± 0.7 1.2 (499 nm)
Thio-NADP
+
2.0 ± 0.2 0.83 (496 nm)
NADPO 3.0 ± 1 1.8 (494 nm)
a
Difference extinction coefficients at the wavelength indicated in
parentheses were calculated by subtracting the absorbance of free
FprA from that extrapolated at infinite ligand concentration.
Oxidation of NADP
+
to its 4-oxo derivative M. de Rosa et al.
4004 FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS
erythrocytes and yeast glucose-6-phosphate dehydrogenase
were all from Roche Diagnostics (Monza, Italy). Horserad-
ish peroxidase was bought from Invitrogen (San Giuliano
Milanese, Milano, Italy). Recombinant M. tuberculosis
wild-type FprA was produced and isolated in two different
molecular forms: without extra residues [10], and with an
N-terminal poly-His extension [5]. The two enzyme forms

were found to be indistinguishable in their functional prop-
erties. The site-directed mutants FprA-H57Q and FprA-
E214A were obtained in poly-histidinylated form only [5].
Recombinant S. oleracea leaf FNR, T. gondii FNR and
P. falciparum FNR were purified as described elsewhere
[11,17,18]. Purified recombinant bovine AdR was a gener-
ous gift of R Bernhardt (Universita
¨
t des Saarlandes, Saar-
bru
¨
cken, Germany). NADP
+
, NADPH, NAD
+
and AMP
were purchased from Sigma-Aldrich (Milano, Italy).
Amplex Red was from Invitrogen. All other chemicals were
of the highest possible grade.
Chromatographic separation of nucleotides
Variable volumes of enzyme reaction mixtures (see below)
were treated with equal volumes of acetonitrile to denatur-
ate and precipitate the protein. After centrifugation at
12 000 g for 10 min, the supernatants were dried, resuspend-
ed in 50 mm ammonium formate, and chromatographed by
a modification of the high-performance ion exchange proce-
dure described in Orr & Blanchard [6]. Using an A
¨
KTA
FPLC apparatus (GE Healthcare), samples were loaded on

a MonoQ HR 5 ⁄ 5 column (1 mL; GE Healthcare), equili-
brated in the above volatile buffer. Nucleotides were separ-
ated at room temperature using a 50–600 mm ammonium
formate gradient in 25 column volumes at a flow rate of
1mLÆmin
)1
. The eluate was monitored continuously by
measuring its absorbance at 254 nm. Fractions containing
NADPO or 3-carboxamide-4-pyridone mononucleotide
(NMNO) were dried under vacuum and stored at ) 20 °C.
MS
MS and MS ⁄ MS data were obtained using an LCQ
ADV MAX ion trap mass spectrometer equipped with an
ESI ion source and controlled by xcalibur software
v.1.3 (Thermo-Finnigan, San Jose, CA, USA). ESI experi-
ments were carried out in positive ion mode under the
following constant instrumental conditions: source voltage
5.0 kV, capillary voltage 10 V, capillary temperature
250 °C, and tube lens voltage 55 V. MS ⁄ MS spectra
obtained by collision-induced dissociation were performed
with an isolation width of 2 Th (m ⁄ z), and the activation
amplitude was around 35% of the ejection RF amplitude
of the instrument, which corresponds to 1.58 V. ESI-MS
of NADPO yielded ions at m ⁄ z 760.18 [MH]
+
and
782.06, amu [MNa]
+
ESI-MS ⁄ MS of the former
ion yielded fragment ions at m ⁄ z 741.74 [MH–H

2
O]
+
,
624.71 [MH–adenine]
+
, 603.66 [MH)4-oxo-nicotinamide–
H
2
O]
+
, 489.71 [MH)4-oxo-nicotinamide-ribose–H
2
O]
+
,
and 329.86 [MH)4-oxo-nicotinamide-ribose-diphosphate–
H
2
O]
+
, amu.
Determination of the absorption and fluorescence
properties of NADPO and NMNO
All absorption and fluorescence emission spectra were recor-
ded on a UV–visible 8453 diode array spectrophotometer
(Agilent, Cernusco sul Naviglio, Milano, Italy) and a Cary
Eclipse spectrofluorimeter (Varian, Leini, Torino, Italy),
respectively. In order to determine its extinction coefficient,
NADPO was quantified on the basis of the amount of the

phosphate released by phosphatase treatment. NADPO at
10–20 nmol was incubated for 1 h with 0.25 units of alka-
line phosphatase at 25 °C in 0.5 m Tris ⁄ HCl (pH 9.0), con-
taining 10 mm MgCl
2
, to hydrolyze the 2¢-phosphate group.
Free phosphate content was determined by the method of
Chen et al. [19]. Known amounts of NADP
+
were used as
controls, verifying the accuracy of the procedure. To isolate
NMNO, c. 10 nmol of NADPO was treated with 2 lgof
phosphodiesterase for 20 min at 25 °Cin20mm Tris ⁄ HCl
(pH 7.7). NMNO was then purified chromatographically as
described above. The absorbance spectrum of NMNO was
recorded both in 20 mm Tris ⁄ HCl (pH 7.7) and after adjust-
ing the pH to c. 1 by the addition of HCl.
Monitoring of the time-course of NADPO
formation catalyzed by various enzymes
The enzymatic conversion of NADP
+
to NADPO was
studied in both aerobic and anaerobic conditions. The aero-
bic reactions were carried out at 25 °C by mixing
50–150 lm enzyme with variable concentrations of
NADP
+
, ranging from 150 lm to 10 mm,in20mm
Hepes ⁄ NaOH (pH 7.0), containing 100 mm NaCl and 10%
glycerol. At different incubation times, 40 lL aliquots were

withdrawn, AMP was added as internal standard, and sam-
ples were analyzed by ion exchange chromatography as des-
cribed above. The amount of NADPO was determined on
the basis of peak integration data provided by unicorn 5
software (GE Healthcare), and the experimentally estimated
e
254
value of 15.6 mm
)1
Æcm
)1
for NADPO. To monitor the
reaction in the absence of molecular oxygen as the oxidant,
c.20lm FprA in the same buffer as above, either in the
absence or in the presence of 140 lm K
3
Fe(CN)
6
, was
placed in an anaerobic cuvette, containing a concentrated
NADP
+
solution in the side arm to yield a final concentra-
tion of 200 lm. After anaerobiosis was established by suc-
cessive cycles of N
2
-flushing and evacuation, reactants were
mixed. Spectral changes were recorded over a period of sev-
eral hours at 20 °C using a UV–visible 8453 diode array
spectrophotometer (Agilent).

M. de Rosa et al. Oxidation of NADP
+
to its 4-oxo derivative
FEBS Journal 274 (2007) 3998–4007 ª 2007 The Authors Journal compilation ª 2007 FEBS 4005
Identification of the reactive oxygen species
produced in the reaction between NADP
+
and
O
2
catalyzed by FprA
Air-equilibrated mixtures of 10 lm FprA and 0.5 mm
NADP
+
were incubated as described in the previous para-
graph in the presence of 0.1 unitÆmL
)1
horseradish peroxi-
dase and 100 lm Amplex Red. Peroxidase-catalyzed
Amplex Red conversion to resorufin was monitored by
measuring the fluorescence emission at 585 nm of the solu-
tion upon excitation at 571 nm. When superoxide dismu-
tase and catalase were present, the concentrations were
0.5 lgÆmL
)1
and 1 lgÆmL
)1
, respectively.
Enzyme activity assays
Assays of the NADPH-dependent catalytic activities of

FprA, spinach leaf (S. oleracea) FNR and T. gondii FNR
were performed under steady-state conditions with different
artificial electron acceptors [iodo-nitro-tetrazolium chloride
(INT) or 2,6-dichloroindophenol] by continuously monitor-
ing the reactions using either an Agilent 8453 diode array
or a Varian Cary 100 double-beam spectrophotometer.
Reaction conditions have been described elsewhere [10]. To
evaluate the inhibitory effect of NADPO, the concentration
of NADPO was varied between 0 and 20 lm, whereas that
of NADPH was independently varied between 0.1 and
10 lm and between 2 and 27 lm, in the case of FprA and
FNRs, respectively. K
i
values of NADPO were determined
by fitting the experimental data points to the theoretical
equation for the competitive inhibition mechanism, using
the nonlinear fitting feature of grafit 5 (Erithacus Soft-
ware Ltd, Horley, Surrey, UK).
Ligand-binding studies
Spectrophotometric titrations of FprA (c.15lm) with
either NADPO, NADP
+
, 2P-AMP or thio-NADP
+
were
performed at 15 °Cin20mm Hepes ⁄ NaOH (pH 7.0), con-
taining 50 mm NaCl, using a Cary 100 double-beam spec-
trophotometer (Varian). The spectra were recorded before
and after successive additions of equal amounts of the nuc-
leotide to the sample and reference cells. Difference spectra

were computed by subtracting the initial spectrum, correc-
ted for dilution, from those recorded after each ligand addi-
tion. K
d
values were computed by fitting the data points to
the theoretical equation for 1 : 1 binding [20], using the
nonlinear fitting feature of grafit 5 (Erithacus Software
Ltd).
Acknowledgements
We are grateful to Rita Bernhardt for providing a
sample of purified recombinant bovine AdR. We also
thank Federico Fischer for his contribution to the ini-
tial part of this research. This work was supported by
grants from Ministero dell’Universita
`
e della Ricerca
of Italy (PRIN 2005) and Fondazione Cariplo, Milano,
Italy.
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