Tài liệu Báo cáo khoa học: The crystal structure of human a-amino-b-carboxymuconatee-semialdehyde decarboxylase in complex with 1,3-dihydroxyacetonephosphate suggests a regulatory link between NAD synthesis and glycolysis ppt

The crystal structure of human
a
-amino-
b
-carboxymuconate-
e
-semialdehyde decarboxylase in complex with
1,3-dihydroxyacetonephosphate suggests a regulatory
link between NAD synthesis and glycolysis
Silvia Garavaglia
1
, Silvia Perozzi
1
, Luca Galeazzi
2
, Nadia Raffaelli
2
and Menico Rizzi
1
1 DiSCAFF Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, University of Piemonte Orientale ‘A. Avogadro’,
Novara, Italy
2 Department of Molecular Pathology and Innovative Therapies, Section of Biochemistry, Universita
`
Politecnica delle Marche, Ancona, Italy
Introduction
In humans, tryptophan at a level that exceeds the basal
requirements for protein and serotonin synthesis is oxi-
datively degraded through the kynurenine pathway,
producing the highly unstable intermediate a-amino-
b-carboxymuconate-e-semialdehyde (ACMS) [1]. As
shown in Fig. 1, ACMS can be either non-enzymati-

cally converted into quinolinic acid (QA), fuelling
NAD biosynthesis, or transformed by the action of
ACMS decarboxylase (ACMSD, also known as picoli-
nate carboxylase; EC 4.1.1.45) into a-aminomuconic
Keywords
cerebral malaria; kynurenine pathway;
metal-dependent amidohydrolase; NAD
biosynthesis; neurological disorders
Correspondence
M. Rizzi, DiSCAFF, University of Piemonte
Orientale, Via Bovio 6, 28100 Novara, Italy
Fax: +39 0321 375821
Tel: +39 0321 375712
E-mail:
Database
The atomic coordinates and structure
factors of hACMSD have been deposited
with the Protein Data Bank (http://
www.rcsb.org) with accession codes 2wm1
and r2wm1, respectively
(Received 1 July 2009, revised 8 September
2009, accepted 10 September 2009)
doi:10.1111/j.1742-4658.2009.07372.x
The enzyme a-amino-b-carboxymuconate-e-semialdehyde decarboxylase
(ACMSD) is a zinc-dependent amidohydrolase that participates in picolinic
acid (PA), quinolinic acid (QA) and NAD homeostasis. Indeed, the enzyme
stands at a branch point of the tryptophan to NAD pathway, and deter-
mines the final fate of the amino acid, i.e. transformation into PA, com-
plete oxidation through the citric acid cycle, or conversion into NAD
through QA synthesis. Both PA and QA are key players in a number of

physiological and pathological conditions, mainly affecting the central ner-
vous system. As their relative concentrations must be tightly controlled,
modulation of ACMSD activity appears to be a promising prospect for the
treatment of neurological disorders, including cerebral malaria. Here we
report the 2.0 A
˚
resolution crystal structure of human ACMSD in complex
with the glycolytic intermediate 1,3-dihydroxyacetonephosphate (DHAP),
refined to an R-factor of 0.19. DHAP, which we discovered to be a potent
enzyme inhibitor, resides in the ligand binding pocket with its phosphate
moiety contacting the catalytically essential zinc ion through mediation of
a solvent molecule. Arg47, Asp291 and Trp191 appear to be the key resi-
dues for DHAP recognition in human ACMSD. Ligand binding induces a
significant conformational change affecting a strictly conserved Trp–Met
couple, and we propose that these residues are involved in controlling
ligand admission into ACMSD. Our data may be used for the design of
inhibitors with potential medical interest, and suggest a regulatory link
between de novo NAD biosynthesis and glycolysis.
Abbreviations
ACMS, a-amino-b-carboxymuconate-e-semialdehyde; ACMSD, a-amino-b-carboxymuconate-e-semialdehyde decarboxylase; DHAP,
1,3-dihydroxyacetonephosphate; PA, picolinic acid; QA, quinolinic acid.
FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6615
acid-e-semialdehyde, which possibly collapses to picoli-
nic acid (PA) [2,3]. Therefore, by competing with the
non-enzymatic synthesis of QA, ACMSD ultimately
controls the metabolic fate of tryptophan catabolism
along the kynurenine pathway, and is a medically rele-
vant enzyme in light of the important roles played by
QA and PA in physiological and pathological condi-
tions. Indeed, QA is not only a key precursor of

NAD, but also a potent neurotoxin that acts by acti-
vating the N-methyl-d-aspartate subtype receptor
for glutamate [4]. QA imbalance was reported to be
associated with a number of neurological disorders,
including a wide range of neuropsychiatric and neuro-
degenerative disease states, such as epilepsy, Alzhei-
mer’s and Huntington’s diseases [5]. Conversely, PA
has been reported to prevent the neurotoxic effects of
increased QA in the rat central nervous system, sug-
gesting that a highly regulated production of these
metabolites is required for normal nervous function
[6]. Consistently, it has recently been shown that the
enzyme is highly expressed in primary adult neurons
but not in SK-N-SH neuroblastoma cells, with a per-
fect correlation between the observed expression profile
and the associated variation in QA and PA levels [7],
and other investigations have clearly demonstrated
that changes in ACMSD activity are readily reflected
by serum and tissue QA levels [8]. Moreover, PA
exhibits important immunomodulatory properties,
being able to stimulate apoptosis [9], to efficiently
interrupt the progress of human HIV-1 infection
in vitro [10], and to activate macrophages in pro-
inflammatory processes [11]. Most recently, abnormally
high brain levels of PA have been reported in a mur-
ine model of cerebral malaria, a frequently fatal com-
plication of Plasmodium falciparum infection; in the
same model, pharmacological reduction of PA levels
was demonstrated to correlate with a better disease
outcome [12,13]. ACMSD is not only present in higher

eukaryotes, but also in some micro-organisms, in
which the enzyme plays a key role in both the trypto-
phan to QA transformation and catabolism of 2-nitro-
benzoic acid [14,15]. Extensive biochemical and
structural characterizations have been carried out on
Pseudomonas fluorescens ACMSD (PfACMSD), lead-
ing to the discovery that the enzyme is a member of
the metal-dependent amidohydrolase superfamily fea-
turing an (a ⁄ b)
8
TIM barrel fold [16–18]. Biochemical
and structural analysis of PfACMSD led to proposal
of a non-oxidative decarboxylation catalytic mecha-
nism, unprecedented amongst known decarboxylases
[18,19]. The gene encoding human ACMSD (hA-
CMSD) was identified few years ago [20], and very
recently the existence of two isoforms originating by
alternative splicing was demonstrated; although
comparably expressed in various organs, only the
hACMSD I isoform was reported to be enzymatically
active and extensively characterized [3]. hACMSD
shares a high degree of sequence identity with
PfACMSD (38%), with strict conservation of all resi-
dues that are proposed to play a key role in catalysis
and are involved in co-ordination of the catalytically
essential zinc ion. As no ACMSD structure with
bound substrate or inhibitor has been reported so far
from any source, understanding of the ACMSD
catalytic mechanism is still incomplete. In light of the
reported ACMSD upregulation in the liver of

streptozotocin-induced diabetic rats and the suppres-
sion of such elevation following insulin injection
[21,22], we decided to investigate the effect of
N
H
CH
2
CH
COOH
NH
2
L-kynurenine
CHO
COOH
COOH
NH
2
Tryptophan
α-amino-β-carboxymuconate-ε-semialdehyde (ACMS)
α-amino-β-carboxymuconate-
ε-semialdehyde decarboxylase
(ACMSD)
CHO
COOH
NH
2
α-aminomuconic acid-
ε-semialdehyde (AMS)
N
COOH

COOH
Quinolinic acid
non enzymatic
NAD
Glutaryl CoA
CO2 + ATP
non enzymatic
N
COOH
Picolinic acid
Fig. 1. The reaction catalyzed by hACMSD in a metabolic context.
ACMS is derived from tryptophan degradation through the kynure-
ine pathway, and, depending on hACMSD activity, has various
metabolic destinies.
Crystal structure of human ACMSD S. Garavaglia et al.
6616 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS
glycolytic intermediates on the enzyme activity.
Interestingly, several phosphorylated glycolytic
intermediates were found to be strong inhibitors of
hACMSD, of which 1,3-dihydroxyacetonephosphate
(DHAP) was the most potent and was therefore
selected for our structural investigation. Our results
provide the first structural image of an ACMSD in a
ligand-bound form, and may be used to assist the struc-
ture-based rational design of enzyme inhibitors with
potential medical interest.
Results and Discussion
Overall quality of the model
The three-dimensional structure of hACMSD in com-
plex with DHAP was solved by molecular replacement

and refined at a resolution of 2.0 A
˚
. The monomer
present in the asymmetric unit contains 332 residues
out of 336, one zinc ion, one DHAP molecule, one glyc-
erol molecule and a total of 216 solvent molecules. No
electron density is present for the last four residues at
the C-terminus. The stereochemistry of the model has
been assessed using the program procheck [23]. Ninety
per cent of the residues fall in the most favoured
regions of the Ramachandran plot, with Asn148 and
His269 falling in disallowed regions. However, for both
residues, the excellent electron density map allowed us
to unambiguously assign the observed conformation.
Residue Pro293 was recognized as the cis conformer.
Overall structure
hACMSD shows a molecular architecture that closely
resembles that described for PfACMSD [18], compris-
ing a distorted (a ⁄ b)
8
barrel domain and a small inser-
tion domain (Fig. 2). hACMSD and PfACMSD can
indeed be superposed with an rmsd of 1.6 A
˚
based on
326 Ca pairs. hACMSD folds into 12 a-helices, 11
b-strands and connecting loops. Residues 14-48 form
the small insertion domain that comprises a short
a-helix and a three-stranded anti-parallel b-sheet; the
remaining protein residues form the (a ⁄ b)

8
barrel
domain and a C-terminal extension that comprises two
short a-helices. Functional hACMSD was previously
reported to be a monomer in solution [3]. Consistently,
one molecule is present in the asymmetric unit in our
crystal, although a dimer can be observed in the crys-
tal lattice by applying the crystallographic two-fold
axis. PfACMSD was reported to be a dimer, with
subunits related by a dyad axis in the crystal, and a
mixture of monomeric and dimeric forms in solution
[18]. Therefore, the available structural data suggest
that the minimal functional unit in the ACMSD
enzyme is a monomer. and the biological significance,
if any, of the loose dimer observed in the crystalline
state remains to be established. The overall structural
organization observed in hACMSD confirms the previ-
ous assignment of the enzyme to the metal-dependent
hydrolase superfamily [17,18], whose members feature
by a structurally conserved TIM a ⁄ b barrel fold [24].
The significant structural conservation observed
between hACMSD and PfACMSD extends to the
peculiar small insertion domain, which may be consid-
ered a unique trait of ACMSDs.
The metal centre and the ligand binding site
The hACMSD active site is located in a crevice on the
protein surface at the C-terminal opening of the b-bar-
rel (Fig. 2), with a Zn
2+
ion occupying the metal

centre and coordinating, with a distorted trigonal
bipyramidal geometry, the strictly conserved residues
His6 (2.0 A
˚
), His8 (2.1 A
˚
), His174 (2.2 A
˚
), Asp291
(2.2 A
˚
) and the water molecule w1 (2.1 A
˚
) (Fig. 3A).
The DHAP binding site protrudes from the metal
N-ter
C-ter
Fig. 2. Ribbon representation of the overall structure of hACMSD.
The (a ⁄ b)
8
barrel domain is colored in light blue and the ACMSD-
specific small insertion domain in blue. The DHAP molecule and
the protein residues involved in metal coordination are shown as
sticks; the Zn
2+
ion and the solvent molecule bridging the metal
centre to the ligand are shown as magenta and cyan spheres,
respectively.
S. Garavaglia et al. Crystal structure of human ACMSD
FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6617

centre into a small pocket delimited by residues
Asp291, Trp191, Met195, Arg47 and the Phe294-
Pro295-Leu296 amino acid stretch (Fig. 3A,B). The
ligand binds in an extended conformation, with its
phosphate moiety located in the proximity of the zinc
ion and with the hydroxymethylene group pointing
toward Pro295. DHAP interacts with the catalytically
essential Zn
2+
through mediation of the metal-coordi-
nating solvent molecule w1, which establishes two
strong hydrogen bonds with the ligand O1 and O1P
atoms at 2.5 and 2.8 A
˚
, respectively. Moreover, the
ligand is engaged in a number of stabilizing interac-
tions with the protein milieu by contacting both pro-
tein residues and solvent molecules. In particular, the
DHAP phosphate moiety establishes a salt bridge with
Arg47 (distance of 3.0 A
˚
), an electrostatic interaction
with the Zn
2+
ion (closest distance of 4.1 A
˚
) and an
extensive network of hydrogen bonds involving a set
of well-ordered solvent molecules. O1P contacts the
solvent molecule w268 (2.8 A

˚
), and O2P interacts with
Trp191 (2.8 A
˚
) and O3P with w51 (2.6 A
˚
), which in
turn contacts w119 (distance of 2.9 A
˚
), which contrib-
utes to fixing the Arg47 orientation by establishing a
hydrogen bond with its NH1 atom (at 2.8 A
˚
). The
DHAP aliphatic chain is sandwiched between Trp191
and Asp291, with its carbonyl oxygen contacting, at a
distance of 2.7 A
˚
, both Asp291 and the solvent mole-
cule w1. Finally, the DHAP hydroxyl group is found
at 3.2 A
˚
from Arg47, whose guanidinium group is held
in the observed conformation by an aromatic stacking
interaction with Phe297.
Implications for catalysis
ACMSD belongs to the metal-dependent amidohydro-
lase superfamily and catalyses a non-oxidative decar-
boxylation reaction through a still not completely
understood mechanism. Extensive biochemical, struc-

tural and spectroscopic investigations, mainly carried
out on PfACMSD, led to the proposal of two possible
alternative catalytic mechanisms [18], whose common
feature involves formation of a tetrahedral intermedi-
ate resulting from the nucleophilic attack of the metal-
bound hydroxyl group onto the substrate, as observed
in other members of the amidohydrolase superfamily
[24,25]. However, as no structure of complexes with
either the substrate, product or inhibitors had been
reported, precise identification of the protein residues
involved in ligand recognition and catalysis remained
elusive.
Although our structural data do not allow us to
discriminate between the two alternative catalytic
His 6
His 8
His 174
Asp 291
w 1
DHAP
Arg 47
Leu 296
Trp 191
Pro 295
w 1
DHAP
w 268
w 119
w 51
Met 195

Phe 294
C 1
A
B
Fig. 3. Close-up view of the active site in hACMSD. (A) The metal
centre. The Zn
2+
ion and the coordinated solvent molecule are
shown as magenta and cyan spheres, respectively. The strictly con-
served protein residues forming the metal coordinative sphere and
the ligand molecule are drawn as balls-and-sticks, with the DHAP
phosphorous in orange. The Zn
2+
coordinative bonds are indicated
by dotted lines, together with the hydrogen bonds established by
DHAP with the metal-coordinating solvent molecule w1. The por-
tion of the 2F
o
)F
c
electron density map covering the DHAP mole-
cule is shown in blue at the 1.2 r level. (B) The DHAP binding site
with crucial protein residues and solvent molecules engaged in
ligand recognition and stabilization are drawn as balls-and-sticks and
spheres, respectively. The major interactions established between
the DHAP inhibitor and the protein milieu are indicated by dotted
lines.
Crystal structure of human ACMSD S. Garavaglia et al.
6618 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS
mechanisms proposed for ACMSD [21], the hA-

CMSD:DHAP complex represents the first structure of
an ACMSD in a ligand-bound form and can be used
to provide insights into catalysis. In particular, the
residues involved in inhibitor binding can be suggested
to be important players for recognition of the physio-
logical substrate ACMS. On the basis of our structure,
we propose that the strictly conserved Asp291 is a fun-
damental residue for catalysis, not only because it con-
tributes to Zn
2+
coordination but also because of its
direct involvement in substrate binding (Fig. 3A,B).
Indeed, as detailed above, Asp291 stabilizes DHAP
through interaction with the inhibitor carbonyl group,
and is therefore likely to demonstrate an equivalent
role on ACMS by possibly recognizing its aldehyde
group (Fig. 1). Our observation is in agreement with
what was observed for PfACMSD, where a significant
increase in the K
M
value was observed when the equiv-
alent residue (Asp294) was mutated to alanine [16].
Another residue emerging as a key molecular determi-
nant for ligand recognition in hACMSD is Arg47
(Fig. 3B). Its guanidinium group contacts both the
phosphate and the hydroxyl moieties present on
DHAP, and appears to be a major contributor to
ACMS recognition by stabilizing interactions with the
negatively charged carboxylic groups and with the
aldehydic portion of the physiological substrate. A

third residue of relevance for efficient ligand recogni-
tion is Phe297. Indeed, in the hACMSD:DHAP struc-
ture, this residue fixes the orientation of Arg47, and is
‘edge-on’ oriented with respect to the DHAP aliphatic
chain with a shortest distance of 3.7 A
˚
from C1
(Fig. 3B). Such a conformation is compatible with
establishment of an aromatic hydrogen bond between
Phe297 and the double bonds present in ACMS.
We carried out a careful comparison between the
hACMSD:DHAP complex and the ligand-free form of
PfACMSD after optimal structural superposition.
Although no major conformational changes affecting
entire domains or extended portions of the protein
structure were detected, a significant change in the ori-
entation of a few residues was observed (Fig. 4).
Indeed, upon ligand binding, a severe conformational
rearrangement takes place for the side chains of Met195
and Trp191, which move unidirectionally toward the
substrate-binding pocket and become engaged with the
bound ligand. Trp191 shows the most pronounced
movement, which consists of a rotation around the v
2
dihedral angle of about 95°, allowing formation of a
hydrogen bond with the DHAP phosphate group. This
switch between two alternative conformations suggests
that the Trp191–Met195 couple is the main active site
gating determinant controlling ligand admission.
Conclusion

hACMSD appears to be an important enzyme control-
ling the cellular levels of QA, PA and NAD. As a
consequence, modulation of hACMSD activity could
be of considerable relevance in certain therapeutic con-
texts [2,26–30]. The various neurological disorders in
which severe imbalance in the kynurenyne pathway is
seen include cerebral malaria [31], where an elevated
level of the pro-inflammatory PA has been proposed to
contribute to the development of this frequently fatal
clinical manifestations of the disease [12,32]. Moreover,
this disease also features a significant depletion of the
NAD+ level [31,33]. We propose that hACMSD inhi-
bition could result in alleviation of cerebral malaria
symptoms by controlling both PA and NAD levels
[7,34–36].
Recently, a direct link between NAD synthesis and
diabetes has been reported [37], suggesting that an
increased NAD level is a desirable condition to combat
the disease. Intriguingly, ACMSD was reported to be
overexpressed in streptozotocin-induced diabetic rats
Trp 191
w 1
DHAP
Met 195
Fig. 4. Conformational changes affecting ACMSD upon ligand bind-
ing. The image was obtained by optimal superposition of the hA-
CMSD:DHAP and PfACMSD:ligand-free structures. Protein residues
are colored in white for hACMSD and in green for PfACMSD, with
the DHAP ligand in yellow; the protein portion shown by a ribbon
representation refers to hACMSD, as do the amino acid numbers.

The alternative conformations of the Trp–Met couple can be
observed; the arrows indicate the unidirectional movement of the
two protein residues upon ligand binding. The dotted line highlights
the hydrogen bond established between the DHAP phosphate
moiety and Trp191 in the ligand-bound form of the enzyme.
S. Garavaglia et al. Crystal structure of human ACMSD
FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6619
[21,22], and insulin injection was observed to suppress
such elevation. Therefore, we propose that inhibition of
hACMSD should be explored as a possible novel thera-
peutic avenue for the treatment of diabetes. In this
respect, the significant enzyme inhibition that was
found to be exerted by intermediates of glycolysis
(Table 1), a pathway imbalanced in diabetes, is intrigu-
ing. Moreover, our structural data reveal that the
enzyme active site is well suited to efficiently bind
DHAP, a central glycolytic intermediate. We are there-
fore tempted to speculate on a possible physiological
relevance of the observed modulation of hACMSD
activity by glycolytic intermediates that would imply a
novel regulatory role of the enzyme in energy metabo-
lism. Indeed, robust aerobic glycolysis requires signifi-
cant NAD+ availability, which could be sustained by a
burst of de novo dinucleotide biosynthesis through tryp-
tophan degradation, an event that implies efficient AC-
MSD inhibition. Therefore, hACMSD would act as a
regulatory link between glycolysis and NAD synthesis.
The structure of hACMSD in complex with DHAP
may used for the design of potent and highly selective
enzyme inhibitors that may prove to be of potential

interest to reduce life-threatening complications of
cerebral malaria, and as an important tool in validat-
ing our proposal of hACMSD as a novel drug target
for the treatment of diabetes and to investigate its
proposed novel regulatory role.
Experimental procedures
Enzyme expression, purification and inhibition
studies
The expression vector pHIL-D2-ACMSDI constructed pre-
viously [3] was used as a template to amplify the ACMSD
gene, resulting in a C-terminal (His6) fusion protein. The
amplicon was cloned into the pHIL-D2 vector and the
NotI-digested construct was used to transform Pichia pas-
toris GS115 cells [3]. Expression of the recombinant pro-
tein was achieved as described previously [3]. Purification
was performed as described previously [3] with the follow-
ing modifications. The hydroxylapatite column was washed
with 130 mm potassium phosphate buffer pH 7.0, 50 mm
NaCl, and the recombinant protein was eluted with
300 mm potassium phosphate buffer pH 7.0, 50 mm NaCl.
The active fractions were pooled and directly applied to a
HisTrap HP column equilibrated in 10 mm potassium
phosphate buffer pH 7.0, 100 mm NaCl. After extensive
washing with the equilibration buffer, elution was per-
formed with a linear gradient of imidazole from 0–0.3 m
in the same buffer. The active fractions were pooled and
diluted 10-fold with 50 mm potassium phosphate buffer,
concentrated by ultrafiltration with a YM30 membrane
(Millipore SpA, Milan, Italy) and used for the crystalliza-
tion trials. Using 400 mL of yeast culture, approximately

1 mg of homogeneuos ACMSD was obtained, with a spe-
cific activity (1.3 unitsÆmg
)1
) and purification index (243-
fold) similar to those of the protein without the His tag,
and a higher overall yield (68%) [3]. hACMSD activity
was determined specrophotometrically, as described previ-
ously [3]. The effect of the tested molecules on the enzyme
activity was investigated in the presence of 5 lm ACMS
substrate.
Crystallization and structure determination
Crystals of hACMSD were obtained using the vapour dif-
fusion technique in hanging drop. A volume of 1 lL of res-
ervoir solution containing 1 mm DHAP, 2% poly(ethylene
glycol) (PEG 400), 0.1 m Na ⁄ Hepes pH 7.5, 2.0 m ammo-
nium sulphate, was mixed with the same amount of a
protein solution at a concentration of 12.7 mgÆmL, and
equilibrated against 500 lL of the reservoir solution, at
20 °C. The crystals grew to a maximum length of 0.2 mm
in approximately 1 week. The presence of DHAP was
found to be essential for crystallization, and we were
unable to grow crystals of hACMSD in a ligand-free form.
For X-ray data collection, crystals were quickly equili-
brated in a solution containing the crystallization buffer
and 20% glycerol as the cryo-protectant, and flash-frozen
at 100 K under a stream of liquid nitrogen. Data up to
2.0 A
˚
resolution were collected using the ID23-2 beamline
of the European Synchrotron Radiation Facility (Grenoble,

France). An X-ray fluorescence scan performed on the hA-
CMSD crystals using the same beamline, clearly indicated
the presence of a Zn metal ion bound to the enzyme. Anal-
ysis of the diffraction data set allowed us to assign the
crystal to the trigonal P3
2
21 or P3
1
21 space group, with
cell dimensions a = b = 86.27 A
˚
and c = 92.84 A
˚
,
containing one molecule per asymmetric unit with a
Table 1. Effect of glycolytic intermediates on the activity of hA-
CMSD. Inhibition values refer to the percentage inhibition exerted
by the indicated metabolite, relative to a reaction carried out in the
absence of any inhibitor (control). Experiments were performed at
37 °C in triplicate in the presence of 5 l
M ACMS substrate.
Metabolite
Metabolite
concentration (m
M)
Inhibition
(%)
Dihydroxyacetonephosphate 1
0.5
0.1

100
100
70
3-Phosphoglycerate 1
0.5
0.1
100
100
25
Glyceraldehyde-3-phosphate 1 40
Phosphoenolpyruvate 1 30
2-Phosphoglycerate 1 20
Crystal structure of human ACMSD S. Garavaglia et al.
6620 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS
corresponding solvent content of 50%. Data were pro-
cessed using the program mosflm [38], and the ccp
4 suite
of programs [39] was used for scaling. Structure deter-
mination for hACMSD was performed by means of
the molecular replacement technique, using the coordi-
nates of a monomer of PfACMSD as the search model
(Protein Data Bank code 2HBV) [18]. The program
amore [40] was used to calculate both cross-rotation
and translation functions in the 10–4 A
˚
resolution
range. The solution of the rotation function was used
to perform the translation function calculations in
both the P3
2

21 and P3
1
21 space groups. A clear solu-
tion was obtained for the former only, allowing us to
unambiguously assign P3
2
21 as the correct space
group. The initial model was subjected to iterative
cycles of crystallographic refinement using the pro-
gram refmac [41], alternated with manual graphic
sessions for model building using the program o [42].
Approximately 7% of the randomly chosen reflections
were excluded from refinement of the structure and
used for the free R-factor calculation [43]. The pro-
gram arp ⁄ warp [44] was used for adding solvent mol-
ecules. When the R-factor decreased to a value of 0.27
at 2.0 A
˚
resolution, inspection of the electron density
map in the enzyme active site clearly revealed the pres-
ence of one molecule of DHAP that was consequently
manually modelled based on both the 2F
o
)F
c
and
F
o
)F
c

electron density maps. The subsequent crystal-
lographic refinement converged to an R-factor and a
free R-factor of 0.19 and 0.24, respectively, with ideal
geometry. Data collection and refinement statistics are
given in Table 2.
Illustrations
Figures were generated by using the program pymol [45]
().
Acknowledgements
The authors would like to thank Dr Franca Rossi
(DiSCAFF, University of Piemonte Orientale, Novara)
for critical reading of the manuscript. This work was
supported by grants from the Regione Piemonte
(Ricerca Scientifica Applicata 2004), Ministero dell’Is-
truzione, dell’Universita
´
e della Ricerca (Bando PRIN
2007) and the Compagnia di San Paolo – IMI (Torino,
Italy) in the context of the Italian Malaria Network.
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Parameter Value
Resolution (A

˚
) 2.0
Observations 63 794
Unique reflections 24 525
Rmerge 0.124
Multiplicity 2.6
Completeness (%) 94
Number of protein atoms 2635
Number of solvent molecules 216
Number of DHAP ⁄ Zn atoms 10 ⁄ 1
Number of glycerol atoms 6
R-factor (%) 19.3
R-free (%) 25.6
rmsd bond lengths (A
˚
) 0.018
rmsd bond angles (°) 1.6
Mean B-factor protein (A
˚
2
) 15.3
Mean B-factor solvent (A
˚
2
) 25.2
Mean B-factor DHAP ⁄ Zn (A
˚
2
) 39.3 ⁄ 10.1
Mean B-factor glycerol atoms (A

˚
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) 25.6
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