Dual Activity of Quinolinate Synthase: Triose Phosphate Isomerase and Dehydration Activities Play Together to Form Quinolinate

Article abstract of DOI:10.1021/acs.biochem.5b00991

Quinolinate synthase (NadA) is an Fe₄S₄ cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The capacity of NadA to use G-3P is due to its previously unknown triose phosphate isomerase activity.

Full text of DOI:10.1021/acs.biochem.5b00991

Subscriber access provided by UNIV OF LETHBRIDGE
Rapid Report
The dual activity of Quinolinate Synthase: Triose Phosphate
Isomerase and dehydration activities play together to form quinolinate
Debora Reichmann, Yohann Couté, and Sandrine Ollagnier de Choudens
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00991 • Publication Date (Web): 12 Oct 2015
Downloaded from http://pubs.acs.org on October 13, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 4
Biochemistry
1
2
3
4
5
6
The dual activity of Quinolinate Synthase: Triose Phosphate Isomer-
ase and dehydration activities play together to form quinolinate
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
,2,3
4,5,6
1,2,3,*
Debora Reichmann , Yohann Couté , Sandrine Ollagnier de Choudens
1 Univ. Grenoble Alpes, iRTSVꢀLCBM, Fꢀ38000 Grenoble, France; 2 CNRS, iRTSVꢀLCBM, Fꢀ38000 Grenoble, France; 3 CEA, iRTSVꢀ
LCBMꢀBiocat, Fꢀ38000 Grenoble, France; 4 Université Grenoble Alpes, iRTSVꢀBGE, Grenoble 38000, France ; 5 CEA, iRTSVꢀBGE,
Grenoble 38000, France ; 6 INSERM, BGE, Grenoble 38000, France.
KEYWORDS NAD, Quinolinate, triose phosphate isomerase, NadA, mechanism
Supporting Information Placeholder
strongly suggesting that the chemistry of QA formation occurring
ABSTRACT: Quinolinate synthase (NadA) is an Fe S clusterꢀ
4
4
2+
13
at the Fe4S
4
cluster takes place on this iron site . Recently, the
containing dehydrating enzyme involved in the synthesis of
quinolinic acid (QA), the universal precursor of the essential
coenzyme nicotinamide adenine dinucleotide (NAD). The
reaction catalyzed by NadA is not well understood and two
mechanisms have been proposed in the literature differing in the
nature of the molecule (DHAP or Gꢀ3P) that condenses with
iminoaspartate (IA) to form QA. In this article, using biochemical
approaches, we demonstrate that DHAP is the triose that
condenses with IA to form QA. The NadA capacity to use Gꢀ3P is
due to its previously unknown triose phosphate isomerase
activity.
crystal structure of the active Fe S ꢀbound NadA from Thermoto-
4
4
14
ga maritima was solved at 1.65 Å resolution . This structure
confirmed the presence of an accessible iron site, coordinated by a
14
water molecule and has allowed the identification of key aminoꢀ
acids in the active site. The structure revealed a long tunnel conꢀ
necting the molecular surface of the enzyme to the H Oꢀ
2
coordinated iron of the cluster where substrates could be accomꢀ
modated.
Nicotinamide adenine dinucleotide (NAD) is an essential and
ubiquitous cofactor involved in a myriad of redox and nonꢀredox
1ꢀ3
reactions . The initial steps in the biosynthesis of NAD vary
considerably between prokaryotes and eukaryotes, although both
pathways involve the common intermediate quinolinic acid (QA).
In most eukaryotic organisms, QA is produced by the oxidative
4
degradation of Lꢀtryptophan by a 5ꢀenzymes pathway . In most
Scheme 1: Mechanisms proposed for quinolinic acid biosynthesis
prokaryotes, including Escherichia coli, and in plants, QA is
generated by a unique condensation reaction between dihydroxyꢀ
acetone phosphate (DHAP) and iminoaspartate (IA). This reaction
requires the concerted actions of two proteins, Lꢀaspartate oxidase
from DHAP or Gꢀ3P and iminoaspartate as published by Sakubaꢀ
15
5
ra et al. (A) and by T. Begley (B) . The iron atom from the
Fe S that functions as a Lewis acid in the reaction is shown (Fe).
4
4
(NadB) that first converts Lꢀaspartate to iminoaspartate and quinꢀ
olinate synthase (NadA) which condenses IA with DHAP to form
QA. Besides these de novo pathways for NAD synthesis, a salꢀ
vage pathway exists in some organisms allowing NAD to be
The reaction catalyzed by NadA is not yet known in terms of
intermediates, but two mechanisms have been proposed for the
5,15
synthesis of QA from DHAP and IA . These mechanisms are
summarized in Scheme 1 where the role of the iron atom from the
Fe S cluster which is available to act as catalyst is also shown.
5
recycled from nicotinic acid (NA) and nicotinamide (Nm) . Howꢀ
ever, some pathogens such as Mycobacterium leprae and Helico-
4
4
6
bacter pylori have been reported to lack this salvage pathway .
Both mechanisms involve the removal of 2 mol of water and 1
16
The different pathways for QA biosynthesis in most prokaryotes
and eukaryotes, combined with the fact that the salvage pathway
is absent in some pathogenic microorganisms makes NadA an
ideal target in the search for specific antibacterial drugs.
mol of inorganic phosphate , but they differ in how the two
substrates are condensed. In mechanism (A), based on early labelꢀ
17
ing studies , condensation occurs between C1 of DHAP and C3
of iminoaspartate, leading to early release of Pi. In mechanism
2
+
(B), in contrast, DHAP is first isomerized into glyceraldehyde 3ꢀ
Asꢀisolated NadA protein binds one oxygenꢀsensitive Fe S
4
4
7
,8
phosphate (Gꢀ3P) which then condenses through its C1 with the
amino group of iminoaspartate, resulting in later release of the
phosphate group. So far, no experimental support has been proꢀ
cluster that is essential for its activity . The cluster is bound by
only three strictly conserved cysteines , reminiscent of dehydraꢀ
9
tases such as aconitase, “RadicalꢀSAM” enzymes or IspG/H
enzymes, where the fourth coordination site is accessible and
5,15
vided for either of these mechanisms . The involvement of Gꢀ
3P as a substrate was initially suggested based on experiments
using partially purified NadA and NadB enzymes and labeled
1
0ꢀ12
occupied by the cognate (co)substrate
. Using a structural
analogue of QA, we demonstrated that the NadA Fe/S cluster also
contains an accessible iron site which is essential for its activity,
18,19
substrates
.
ACS Paragon Plus Environment

Biochemistry
Page 2 of 4
To gain insight into NadA reaction mechanism, in particular to
E. coli BSA displayed no activity at all (data not shown). Since
the Fe S cluster of NadA converts to an Fe S cluster under
aerobic conditions, the isomerization experiment was repeated
anaerobically. No significant change in activity was observed
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
determine which mechanism (A or B) is more likely, we comꢀ
pared Gꢀ3P vs DHAP as substrate in the presence of IA for QA
formation using purified proteins. Through biochemical methods
we demonstrated that purified NadA from different organisms has
a triose phosphate isomerase (TPI) activity catalyzing the reversiꢀ
ble isomerization of Gꢀ3P into DHAP in an Fe/S dependent manꢀ
ner. However, only DHAP can condense with IA to produce QA.
Thus, our results suggest that mechanism A is used for QA forꢀ
mation. Physiological explanations for this dual activity are disꢀ
cussed.
4
4
2 2
(0.31±0.002
(aerobiosis)
vs
0.33±0.01
(anaerobiosis)
µmol/min/mg for DHAP production and 0.028±0.003 (aerobiosis)
vs 0.022±0.006 (anaerobiosis) µmol/min/mg for Gꢀ3P
production), suggesting that the Fe S cluster is not important for
4
4
the TPI activity of NadA (Fig. S3A in SI).
10000
120
A
B
1000
100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
First, we measured quinolinate synthase activity in vitro by
1
00
10
1
mixing purified NadA, NadB and Lꢀaspartate (precursor of IA)
80
ꢀ
1
with either DHAP or Gꢀ3P. A kcat of 9.9 min (Table 1) was
6
4
0
0
determined in the presence of Gꢀ3P and IA, and a similar k
cat
ꢀ
1
(13.9 min ) was determined with DHAP and IA. The affinity of
0 ., 1
NadA for both triose phosphate compounds was also very close,
with a KM value of 0.64±0.12 mM for Gꢀ3P and 0.74±0.08 mM
for DHAP. Thus, catalytic efficiencies were very similar (Table
0 ., 01
20
0
0 ., 001
TPI
NadA Ec
NadA Tm
0
0.5
1
2
5
PGA (mM)
1
). In conclusion, both triose phosphates can be used to form QA,
with Gꢀ3P performing as well as DHAP in the presence of IA.
These results confirmed those of labeling experiments performed
with NadA and NadB enriched extracts supplied with Lꢀaspartate
Figure 1. TPI activity of commercial TPI, E. coli NadA and T.
maritima NadA. Rabbit muscle TPI (1ꢀ5 nM), E. coli NadA (5ꢀ10
µM) or T. maritima NadA (20 µM) were mixed with NADH (0.24
mM), Gꢀ3P (4 mM) and GPDH (1 µM) in 0.1 M TEA pH 7.6 in
aerobic conditions at 37 °C (maintained using a water bath). The
initial rate of the reaction (black) was monitored at 340 nm for
100 sec with a spectrophotometer. For the reverse reaction (light
gray) proteins were added to NAD (1 mM), potassium arsenate
1
4
14
and CꢀDHAP or CꢀGꢀ3P, where similar amounts of radioactive
18
QA were detected .
ꢀ
1
ꢀ1 ꢀ1
kcat (min )
13.9
K (mM)
0.74 ± 0.08
0.64 ± 0.12
keff (M . s )
M
DHAP
Gꢀ3P
313
258
(
0.5 mM), DHAP (4 mM) and GAPDH (0.7 µM). For inhibition
9.9
studies PGA (1 mM) was added (Gꢀ3P substrate: dark gray;
DHAP substrate: white) (n≥3). (B) Inhibition of E. coli NadA
QA-formation by titrated PGA. E. coli NadA (10 µM) was
incubated anaerobically for 20 min at 37 °C with DHAP (2 mM)
(black) or Gꢀ3P (white), OAA (5 mM), AS (10 mM) and PGA (0ꢀ
5 mM) in 0.1 M Hepes pH 7.5, 50 mM KCl (n=13).
Table 1. Kinetic parameters determined for DHAP and G-3P.
E. coli NadA (10 µM) was incubated for 20 min at 37°C with 10
mM Lꢀaspartate, 25 mM fumarate, 10 µM NadB, and DHAP or
Gꢀ3P at concentrations ranging from 0 to 8 mM in 0.1 M Hepes
pH 7.5, 0.05 M KCl under anaerobic conditions (n≥4).
However, when the experiment was repeated using apoꢀNadA, a
0% drop in activity was observed (0.5 vs 0.89 µmol/min/mg for
DHAP formation and 0.04 vs 0.09 µmol/min/mg for Gꢀ3P
formation) (Fig. S3B in SI), indicating that the Fe/S cluster of
NadA (both Fe S and Fe S ) might play a significant structural
Gꢀ3P and DHAP are triose phosphate molecules that can
interconvert within cells thanks to the TPI activity of triose
phosphate isomerase enzymes. TPI are extremely efficient cellular
enzymes which all favor DHAP formation. TPI from eukaryotes
5
5
ꢀ1
4 4 2 2
have a kcat of 2.5ꢀ5.1 x 10 min from Gꢀ3P to DHAP and a k of
cat
4
ꢀ1
20,21
role in NadA which could affect the TPI activity. To eliminate
any possible contamination, we performed two experiments: 1) a
mass spectrometryꢀbased proteomics analysis of purified E. coli
NadA; 2) measurement of TPI activity with purified NadA from a
thermophilic organism, T. maritima. Purification of this enzyme
involved a heating step. The E. coli TPI, TpiA, was not detected
by mass spectrometry analysis of purified NadA (Table S1 and
Figure S6 in SI) and T. maritima NadA was shown to display TPI
activity in the same order of magnitude as that for E. coli NadA,
once again displaying a thermodynamically favorable conversion
direction from Gꢀ3P to DHAP (0.24 µmol/min/mg against 0.01
µmol/min/mg for Gꢀ3P production) (Figure 1). Taken together,
these results demonstrate that NadA is endowed with a TPI
activity, which could explain why it can use both Gꢀ3P and
DHAP along with IA as substrates to produce QA. This activity
makes sense in the context of cellular metabolism where both
DHAP and Gꢀ3P play key roles. During glycolysis fructose 1,6
bisphosphate is converted by fructose 1,6 bisphosphate aldolase
into DHAP and Gꢀ3P. Gꢀ3P is then used for glycolysis (glucose
catabolism) while DHAP is used for neoglucogenesis (glucose
synthesis) (Fig. S4 in SI). The ability of NadA to use both trioses
enables it to ensure the synthesis of QA, and thus NAD even if
one of the two trioses is not available. These results revealed a
new activity of NadA, but they do not provide any information as
to which molecule, DHAP or Gꢀ3P, condenses with IA to form
QA, i.e, whether mechanism A or B is correct. Therefore, in
subsequent experiments we used phosphoglycolic acid (PGA), an
2
.5ꢀ5.2 x 10 min for the reverse reaction
. The TPI from
3
ꢀ
Escherichia coli, TpiA, has a similar activity (k = 6.1 x 10 min
cat
1
22
from Gꢀ3P to DHAP) . We investigated whether NadA is
endowed with TPI activity based on spectrophotometric assays
involving NAD/NADHꢀdependent reactions. For conversion of
Gꢀ3P to DHAP, DHAP production was measured by tracking the
consumption of NADH by glycerolꢀ3ꢀphosphate dehydrogenase
(
GPDH) (Fig. S1 in Supporting Information (SI)). The reverse
+
reaction was monitored using NAD ꢀdependent glyceraldehydeꢀ3ꢀ
phosphate dehydrogenase (GAPDH) (Fig. S1 in SI). We
23
measured the TPI activity of E. coli NadA under aerobic
conditions against a positive control, commercial rabbit muscle
TPI. As shown in Figure 1, NadA can convert Gꢀ3P into DHAP
and back again. Like all TPI enzymes, it is more efficient in the
direction of DHAP production (1.2±0.05 µmol/min/mg against
0
.084±0.02 µmol/min/mg for Gꢀ3P production). Therefore, NadA
exhibits TPI activity in vitro, although this activity is much less
potent than that of the rabbit muscle TPI (10 000ꢀfold lower)
(Figure 1). Interconversion between DHAP and Gꢀ3P is enzymeꢀ
dependent since with the reaction time we used, practically no
DHAP or Gꢀ3P production could be detected using Gꢀ3P and
DHAP as sole components of the reaction mixture, respectively.
Indeed, with Gꢀ3P 4.6% conversion was measured, while
incubation of DHAP resulted in 0.6% conversion after 30 min.
(Fig. S2 in SI). The TPI activity measured with E. coli NadA was
specific, as E. coli ferredoxin, an Fe S protein, and commercial
2
2
2
ACS Paragon Plus Environment

Page 3 of 4
Biochemistry
2
4
inhibitor of TPI activity . PGA is a transition state analog of TPI
which is one atom shorter than the substrate. It binds tightly to the
protein due to its structural and charge similarities to the putative
cisꢀenediolateꢀlike transition state structure of the TPI catalytic
Funding Sources
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SODC thanks the French National Research Agency (Agence
Nationale pour la Recherche) for the NADBIO contract (ANRꢀ
12ꢀBS07ꢀ0018ꢀ01) and acknowledges the partial financial support
from the ARCANE Labex (ANRꢀ11ꢀLABXꢀ0003ꢀ01). YC thanks
the ProFi Infrastructure (ANRꢀ10ꢀINBSꢀ08ꢀ01).
25
24
active site and provides good inhibition at 1 mM . First, we
checked that PGA inhibits NadA TPI activity. In the presence of 1
mM PGA, the TPI activity for both E. coli and T. maritima NadA
was reduced to 10% (Figure 1A). Commercial rabbit TPI was
inhibited by PGA to a similar extent (Figure 1A). Thus, PGA can
be used at 1 mM to significantly inhibit the TPI activity of NadA
and therefore should allow us to determine whether this enzyme
uses DHAP or Gꢀ3P to condense with IA. Using DHAP and IA as
substrates, QA production was unchanged with 1 mM PGA, while
it was slightly affected with 2 to 5 mM PGA (3% and 5%
reduction respectively) (Figure 1B). In contrast, when NadA was
mixed with Gꢀ3P and IA, a 60% drop in activity was observed in
the presence of 1 mM PGA (90% with 5 mM PGA). Similar
effects were observed with the thermophilic NadA enzyme (Fig.
S5 in SI). All these results show that if Gꢀ3P is not converted to
DHAP no QA can be produced, demonstrating that DHAP is the
triose which condenses with IA to generate QA. These results
raised the following question: how QA is produced from DHAP
and IA in the presence of PGA? In other words, is the same active
site used for TPI and quinolinate synthase activities? If two active
sites exist (AS1 and AS2), in AS1 (TPI active site) the PGA
molecule would block Gꢀ3P binding and isomerization into
DHAP, thus hindering QA formation in AS2. In contrast, DHAP,
even though it cannot be converted to Gꢀ3P at AS1, could react
directly with IA in AS2 to produce QA (Scheme S1ꢀA). In the
case of a single active site (AS) for both activities, PGA would
prevent all trioses binding and isomerization. However, the
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank M. Fontecave (Collège de France) for scientific
discussions.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
REFERENCES
(1) Frey, P.; Hegeman, A. D. (2007) Oxford University Press, Oxford.
(2) Belenky, P.; Bogan, K. L.; Brenner, C. (2007) Trends. Biochem. Sci.
3
2, 12ꢀ19.
(
(
3) Pollak, N.; Dolle, C.; Ziegler, M. (2007) Biochem. J. 402, 205ꢀ218.
4) Foster, J. W.; Moat, A. G. (1980) Microbiol. Rev. 44, 83ꢀ105.
(5) Begley, T. P.; Kinsland, C.; Mehl, R. A.; Osterman, A.; Dorrestein, P.
(2001) Vitam. Horm. 61, 103ꢀ119.
(6) Gerdes, S. Y.; Scholle, M. D.; D'Souza, M.; Bernal, A.; Baev, M. V.;
Farrell, M.; Kurnasov, O. V.; Daugherty, M. D.; Mseeh, F.; Polanuyer, B.
M.; Campbell, J. W.; Anantha, S.; Shatalin, K. Y.; Chowdhury, S. A.;
Fonstein, M. Y.; Osterman, A. L. (2002) J. Bacteriol. 184, 4555ꢀ4572.
(
7) Ollagnierꢀde Choudens, S.; Loiseau, L.; Sanakis, Y.; Barras, F.;
Fontecave, M. (2005) FEBS lett. 579, 3737ꢀ3743.
8) Cicchillo, R. M.; Tu, L.; Stromberg, J. A.; Hoffart, L. M.; Krebs, C.;
(
Booker, S. J. (2005) J. Am. Chem. Soc. 127, 7310ꢀ7311.
(9) Rousset, C.; Fontecave, M.; Ollagnier de Choudens, S. (2008) FEBS
Lett. 582, 2937ꢀ2944.
(10) Vey, J. L.; Drennan, C. L. (2011) Chem. Rev. 111, 2487ꢀ2506.
(11) Beinert, H.; Kennedy, M. C.; Stout, C. D. (1996) Chem. Rev. 96,
condensation product of DHAP with IA (Int ) could displace
A
2335ꢀ2335.
PGA to allow QA formation (Scheme S1ꢀB). This situation could
not occur with Gꢀ3P which does not condense with IA. Formation
of IntA from DHAP and IA is favored for two reasons: i)
condensation of DHAP with IA is thermodynamically favorable
with spontaneous release of inorganic phosphate and ii) some
(12) AhrensꢀBotzong, A.; Janthawornpong, K.; Wolny, J. A.; Tambou, E.
N.; Rohmer, M.; Krasutsky, S.; Poulter, C. D.; Schunemann, V.; Seemann,
M. (2011) Angew. Chem. Int. Ed. Engl. 50, 11976ꢀ11979.
(13) Chan, A.; Clemancey, M.; Mouesca, J. M.; Amara, P.; Hamelin, O.;
Latour, J. M.; Ollagnier de Choudens, S. (2012) Angew. Chem. Int. Ed.
Engl. 51, 7711ꢀ7714.
14,15
crystallographic data support early phosphate departure
. Inꢀ
(14) Cherrier, M. V.; Chan, A.; Darnault, C.; Reichmann, D.; Amara, P.;
deed, NadA 3Dꢀstructures show that there is no room in the active
site to accommodate a condensation product on which the phosꢀ
phate group from DHAP is still present.
Ollagnier de Choudens, S.; FontecillaꢀCamps, J. C. (2014) J. Am. Chem.
Soc. 136, 5253ꢀ5256.
(15) Sakuraba, H.; Tsuge, H.; Yoneda, K.; Katunuma, N.; Ohshima, T.
In conclusion, this study demonstrated for the first time that NadA
exhibits a TPI activity with similar properties to other TPI enꢀ
zymes. This activity was exploited to determine the mechanism
used by NadA to produce QA. The formal demonstration that
only DHAP reacts with IA to form QA discriminates between the
two proposed mechanisms in favor of mechanism (A) (Scheme
(
2005) J. Biol. Chem. 280, 26645ꢀ26648.
(16) Chandler, J. L.; Gholson, R. K. (1972) Biochem. Biophys. Acta. 264,
311ꢀ318.
(17) Nasu, S.; Wicks, F. D.; Gholson, R. K. (1982) J. Biol. Chem. 257,
626ꢀ632.
(
18) Suzuki, N.; Carlson, J.; Griffith, G.; Gholson, R. K. (1973) Biochim.
Biophys. Acta 304, 309ꢀ315.
19) Chandler, J. L.; Gholson, R. K.; Scott, T. A. (1970) Biochim.
Biophys. Acta 222, 523ꢀ526.
20) Krietsch, W. K.; Pentchev, P. G.; Klingenburg, H.; Hofstatter, T.;
Bucher, T. (1970) Eur. J. Biochem. 14, 289ꢀ300.
21) Straus, D.; Gilbert, W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,
1
), with early phosphate release, which is supported by crystalloꢀ
(
graphic data. This discovery paves the way for mechanistic
investigations of NadA, in particular to identify intermediates and
design inhibitors.
(
(
ASSOCIATED CONTENT
2014ꢀ2018.
(22) Haley, R.; Fruchtl, M.; Brune, E. M.; Ataai, M.; Henry, R.; Beitle, R.
Supporting Information
(2014) J. Biotechnol. 188, 48ꢀ52.
(23) Lambeir, A. M.; Opperdoes, F. R.; Wierenga, R. K. (1987) Eur. J.
Material and methods and additional figures. This material is
available free of charge via the Internet at http://pubs.acs.org.
Biochem. 168, 69ꢀ74.
24) Wolfenden, R. (1969) Nature, 223, 704ꢀ705.
(
(25) Wierenga, R. K.; Kapetaniou, E. G.; Venkatesan, R. (2010) Cell.
Mol. Life Sci. 67, 3961ꢀ3982.
AUTHOR INFORMATION
Corresponding Author
Telephone: +33438789122; Email: sollagnier@cea.fr
3
ACS Paragon Plus Environment

Biochemistry
Page 4 of 4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For Table of Contents Use Only
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
ACS Paragon Plus Environment