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Eur. J. Biochem. 271, 2765–2772 (2004) Ó FEBS 2004
doi:10.1111/j.1432-1033.2004.04205.x
The crystal structure of glucose-6-phosphate isomerase from
Leishmania mexicana reveals novel active site features
Artur T. Cordeiro1, Paul A. M. Michels2, Luiz F. Delboni3 and Otávio H. Thiemann1
1
Laboratory of Protein Crystallography and Structural Biology, Physics Institute of São Carlos, University of São Paulo,
São Carlos-SP, Brazil; 2Research Unit for Tropical Diseases and Laboratory of Biochemistry, Christian de Duve Institute of
Cellular Pathology, Brussels, Belgium; 3Pontificia Universidade Católica de Minas Gerais, Poços de Caldas-MG, Brazil
Glucose-6-phosphate isomerase catalyzes the reversible
aldose-ketose isomerization of D-glucose-6-phosphate to
D-fructose-6-phosphate in glycolysis and gluconeogenesis,
and in the recycling of hexose-6-phosphate in the pentose
phosphate pathway. The unicellular protozoans, Trypanosoma brucei, T. cruzi and Leishmania spp., of the order
Kinetoplastida are important human parasites responsible
for African sleeping sickness, Chagas’ disease and leishmaniases, respectively. In these parasites, glycolysis is an
important (and in some cases the only) metabolic pathway
for ATP supply. The first seven of the 10 enzymes that
participate in glycolysis, as well as an important fraction
of the enzymes of the pentose phosphate pathway, are
compartmentalized in peroxisome-like organelles called
glycosomes. The dependence of the parasites on glycolysis,
the importance of the pentose phosphate pathway in
defense against oxidative stress, and the unique compartmentalization of these pathways, point to the enzymes
contained in the glycosome as potential targets for drug
design. The present report describes the first crystallographic structure of a parasite (Leishmania mexicana)
glucose-6-phosphate isomerase. A comparison of the
atomic structure of L. mexicana, human and other
mammalian PGIs, which highlights unique features of the
parasite’s enzyme, is presented.
Leishmania mexicana is a human protozoan pathogen
belonging to the order Kinetoplastida [1,2]. Among the
kinetoplastid organisms, several human parasites are present, including Trypanosoma brucei, T. cruzi and various
Leishmania species that are responsible for diseases such as
African sleeping sickness, Chagas’ disease and leishmaniases, respectively, causing serious health problems in
tropical and subtropical areas, which, in several cases, are
fatal if left untreated. This scenario is aggravated by a lack
of effective, available drugs for the treatment of infected
individuals, and the reports of drug-resistant parasite
strains. Leishmania infection may lead to disorders that
can manifest themselves in three different clinical forms –
cutaneous, visceral and mucocutaneous leishmaniasis –
depending on the Leishmania species involved. The actual
treatment for leishmaniasis is based mainly on antimonial
compounds that are of low specificity and cause undesirable
side-effects [1,2].
Glycolysis is an important, and in some cases the only,
metabolic pathway for the ATP supply of these parasites.
The first seven of the 10 enzymes that participate in
glycolysis are compartmentalized in peroxisome-like organelles called glycosomes [3], a characteristic of all members of
the Kinetoplastida order. A consequence of this organellar
localization is that the kinetoplastid glycolytic enzymes
differ in many kinetic and structural properties from their
counterparts in other organisms, and that the flux through
the pathway is regulated in a different manner [2,3]. Not
only glycolysis is found in glycosomes; also found is a
significant fraction of many enzymes of the pentose
phosphate pathway [4,5], which uses sugars for the formation of D-ribose-5-phosphate for nucleotide synthesis and
NADPH, for biosynthetic processes, and for defense against
oxidant stress. This process is also very important for the
trypanosomes and leishmanias, particularly to combat
oxidative attack by the host. Therefore, both the glycolytic
and pentose phosphate pathways have been indicated as
promising drug targets [2,6,7].
Glucose-6-phosphate isomerase (often still called by its
old name, phosphoglucose isomerase; PGI) is the second
enzyme in glycolysis and catalyzes the reversible aldoseketose isomerization of D-glucose 6-phosphate (D-Glc6P) to
D-fructose 6-phosphate (D-Fru6P). It is also an enzymatic
link between glycolysis and the pentose phosphate pathway.
Correspondence to O. H. Thiemann, Laboratory of Protein Crystallography and Structural Biology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo,
Avenue Trabalhador Sãocarlense 400, PO Box 369, 13566–590, São
Carlos-SP, Brazil. Fax: + 55 16 273 9881, Tel.: + 55 16 273 8089,
E-mail: [email protected]
Abbreviations: D-Fru6P, D-fructose-6-phosphate; D-Glc6P, D-glucose6-phosphate; dPGI-Lm, N-terminally deleted glucose-6-phosphate
isomerase from Leishmania mexicana; PGI, glucose-6-phosphate
isomerase; PGI-Lm, glucose-6-phosphate isomerase from
Leishmania mexicana.
Enzyme: glucose-6-phosphate isomerase (E.C. 5.3.1.9).
Note: The PDB ID code for the solution structures of
Leishmania mexicana glucose-6-phosphate isomerase full-length
PGI-Lm is 1Q5O and of the form with the 48 residues deleted from
its N-terminus (dPGI-Lm), 1Q1I.
(Received 3 December 2003, revised 31 March 2004,
accepted 6 May 2004)
Keywords: Leishmania; phosphoglucose isomerase; glycolysis; substrate–enzyme; human PGI.
Ó FEBS 2004
2766 A. T. Cordeiro et al. (Eur. J. Biochem. 271)
In the pentose phosphate pathway, PGI recycles one of the
products (D-Fru6P) back into the substrate (D-Glc6P) for
glucose-6-phosphate dehydrogenase, representing the initial
step of the pathway.
The pentose phosphate pathway appears to have a dual
localization in both Leishmania spp. and T. brucei, as
several of its enzymes have been shown to be present in both
the cytosol and the glycosomes [4,5]. Whereas most
glycolytic enzymes are entirely or predominantly present
inside the organelles, PGI, the enzyme shared with the
pentose phosphate pathway, is indeed found in both
compartments, although in a ratio that differs between
Kinetoplastida species. In bloodstream-form T. brucei,
most ( 85%) PGI resides in the glycosomes, but in
cultured L. mexicana promastigotes (representative of the
insect-infective stage), PGI activity was detected mainly in
the cytosol, with the remainder (less than 10%) associated
with the glycosome [8,9]. This is consistent with a higher
pentose phosphate pathway activity and a lower glycolytic
activity in promastigotes when compared to the bloodstream form of T. brucei [10–12]. The involvement of PGI in
two important pathways of Leishmania metabolism may
make it interesting for drug targeting. Recently, a 50%
growth inhibition in bloodstream-form T. brucei was
observed as a consequence of decreasing the level of PGI
by RNA interference. This result is indicative of the central
role of PGI in the parasite metabolism [13].
In this report we present the atomic structure of
L. mexicana PGI (PGI-Lm) obtained by X-ray diffraction
techniques. The comparison of this structure with the
available mammalian PGI structures allowed the identification of significant differences between the enzyme of the
parasite and its human homologue, which may be exploited
in future drug design.
Experimental procedures
Crystallization data collection and structure
determination
Two different constructs of the PGI gene from L. mexicana
have been expressed in Escherichia coli BL21(DE3) and
purified to homogeneity [14]. The two forms correspond to
the entire 604 amino acid PGI sequence (PGI-Lm) and a
polypeptide from which the N-terminal 47 amino acid
residues were deleted (dPGI-Lm), respectively. The
N-terminal deletion from L. mexicana PGI does not
interfere with the catalytic process. The N-terminal extension is believed to represent an unorganized structure,
possibly related to the glycosomal localization of the protein
[8,9,14], and could be interfering in the crystallization
process. Both forms of the bacterially expressed L. mexicana PGI (PGI-Lm and dPGI-Lm) were successfully crystallized by the hanging drop vapor-diffusion technique, but the
dPGI-Lm crystals presented a better intrinsic order [14].
A complete PGI-Lm dataset of 228 frames was collected
at 100 K from a single crystal grown under conditions
previously reported [14], using a RIGAKU X-ray source
and a MAR345 image plate detector. The crystalto-detector distance was set to 250 mm and each frame
was exposed for 4 min with a phi oscillation of 0.75°. The
dataset was processed using DENZO and SCALEPACK [15].
The molecular replacement solution obtained in the
previous work was used as an initial model in the
refinement procedure. Crystals of dPGI-Lm were soaked
for 5 min in cryoprotectant solution consisting of the
reservoir solution and 17.5% methane pentanediol (w/v)
containing 3.0 mM D-Fru6P and then flash-frozen in liquid
nitrogen. A complete dataset of 140 frames was collected
at 100 K for the dPGI-Lm at the X25 beam line of the
Brookhaven National Laboratory. This beam line is
equipped with a Q315 ccd detector, which allowed the
collection of reflections at a 2.3 Å resolution limit with the
detector placed 325 mm from the crystal. Each frame was
exposed for 15 s with a phi oscillation of 0.75°. The frames
were integrated using MOSFLM [16] and the reflections were
scaled using SCALA from the CCP4 package [17]. The
refined PGI-Lm structure, collected using the RIGAKU
X-ray source, was used as initial model in the refinement
of the dPGI-Lm.
Structure refinement
All refinement procedures were performed using the CNS
program [18], except for a final TLS parameters refinement performed with REFMAC5 from the CCP4 package
[17]. The first structure refined was the PGI-Lm. One
monomer of the previously reported solution, obtained
using the AMORE program [19], was submitted to a rigid
body routine with data ranging between 30 and 3 Å
resolution. Several cycles of simulated annealing, using the
maximum likelihood method, were performed, followed
by coordinate and B-factor refinement using all data up to
the 2.6 Å resolution border. Local corrections and
N-terminal amino acid residue additions were performed using the O program [20] while inspecting the
2rA|Fo|-D|Fc| map and the m|Fo|-D|Fc| difference map.
Water molecules were introduced running the Ôwater-pickÕ
script of CNS. Finally, all model atoms were used to define
a single group for refinement of TLS parameters with
REFMAC5 [17].
The dPGI-Lm data were refined following the same
procedures adopted for the PGI-Lm. The water molecules
and additional N-terminal residues from the refined PGILm structure were removed from dPGI-Lm. The modified
dPGI-Lm polypeptide chain was used as an initial model for
the rigid body refinement. Prior to water addition, D-Fru6P
was placed into the only large electron density cloud
observed at the difference map contoured at 5 s. The correct
orientation of the phosphate group, relative to the protein
residues involved in its coordination, was driven by
structural similarity to the previously reported rabbit PGI
in complex with D-Fru6P [21]. Additional information of
the D-Fru6P conformation was obtained by observing the
2rA|Fo|-D|Fc| map contoured at 1 s. Water molecules were
added using the Ôwater-pickÕ script of CNS with the same
parameters as applied to the PGI-Lm. A single group
containing all atoms from the model was used for refinement of TLS parameters with REFMAC5 [17].
Structure comparison
Superposition and root mean square (r.m.s.) calculations
were performed using the Ca atoms and the SWISS PDB
Ó FEBS 2004
Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2767
program [22]. D-Fru6P and dPGI-Lm side-chain
contacts were analyzed by using the LIGPLOT program [23].
Figures were prepared using the PYMOL program [24].
VIEWER
Results and discussion
Structure refinement
The previously reported molecular replacement solution
[14] was characterized as containing one homodimer
molecule per asymmetric unit in the P61 space group.
The processing of the data described in this study indicate
that the noncrystallographic twofold axe, which relates the
monomers in the asymmetric unit, described previously, is
coincident to a real crystal axe from the higher-symmetry
P6122 space group. The choice of P6122 crystal space
group reduced the asymmetric unit content to a single
monomer. The first Leishmania PGI structure refined,
PGI-Lm (pdb code, 1Q5O), had a total of 216 water
molecules added, resulting in an Rfactor value of 19.5%
and an Rfree value of 24.9%. The refinement of dPGILm-D-Fru6P was concluded with Rfactor and Rfree values
of 22.0 and 25.9%, respectively. A total of 180 water
molecules and one D-Fru6P are present in the final dPGILm/D-Fru6P model (pdb code, 1Q1I). The refinement of
TLS parameters contributed to a decrease of 2% in final
R and Rfree values for both molecules. Additional
information of both refined structures is presented in
Table 1.
Description of structures
The L. mexicana PGI structure is a homodimer and the
monomer subunit is composed of a large and a small a/b
sandwich domain and has an extended C-terminal
segment (comprising two a-helices) that embraces the
other monomer (Fig. 1). There are two catalytic sites per
dimer molecule; the catalytic sites are located in the dimer
interface formed by adjacent monomers. The PGI-Lm has
an overall fold similar to those described for rabbit,
human and pig PGIs [25–27]. The main difference is the
presence of an N-terminal sequence in PGI-Lm, which
may be involved in the functioning of the enzyme inside
the glycosome, as discussed previously [8,9,14]. For the
first 44 and the last residue – Leu605 – of PGI-Lm
(Fig. 2), no electron density was distinguishable in the
2rA|Fo|-D|Fc| map of both chains of the homodimer. The
absence of continuous electron density for residues 1–44
indicates that the N-terminal sequence is not ordered.
Based on the electron density map and sequence alignment, four additional residues (Val45 to Ser48) could be
added to that region of PGI-Lm (Fig. 2). The superposition of the PGI-Lm and the N-terminally deleted
dPGI-Lm resulted in an r.m.s. of 0.4 Å. The main
difference between the two structures is found in a
10-residue loop (of amino acids Gly433 to Ala442) in
which the catalytic His441 is located. Owing to the
improved electron density map and higher-resolution data
obtained with dPGI-Lm crystals, it was possible to
identify the correct Ca trace for this 10-residue loop.
Differences vs. mammalian PGIs
Table 1. Statistics for data collection and refinement. dPGI-Lm,
N-terminally deleted glucose-6-phosphate isomerase from Leishmania
mexicana; PGI-Lm, glucose-6-phosphate isomerase from Leishmania mexicana.
Structure
Data collection
Space group
Cell dimension
a, b and c (Å)
a, b and c (°)
Resolution range (Å)
Unique reflections
Redundancy
Completeness (last shell) (%)
R-sym (%)
Refinement
Number of atoms
Protein
Heteroatoms
Solvent
Used reflections
R-factor
R-free
Rms
bond
angle
<B-factor> (Å2)
PGI-Lm
dPGI-Lm
P6122
P6122
85.74, 85.74
and 350.43
90, 90 and 120
30–2.6
24333
6.7
98.5 (98.8)
5.8
85.13, 85.13
and 350.09
90, 90 and 120
74.53–2.35
32626
7.1
100 (100)
6.1
8607
0
216
23091
19.5
24.9
8649
32
180
30902
22.0
25.9
0.022
1.83
30.9
0.018
1.69
32.8
A significant difference in Ca-r.m.s. can be observed in part
of the small domain of Leishmania PGI compared with its
Fig. 1. Cartoon representation of Leishmania mexicana glucose-6phosphate isomerase (PGI-Lm). The native enzyme is a homodimer
with the monomer subunit formed by two a/b sandwich domains
(large and small domains) and a C-terminal a-helix segment that
embraces the adjacent monomer. The catalytic residues are distributed
among the small domain and C-terminal segment from one monomer,
and at the large domain from the other monomer.
2768 A. T. Cordeiro et al. (Eur. J. Biochem. 271)
Ó FEBS 2004
Fig. 2. Structure-based alignment of glucose-6-phosphate isomerase (PGI) sequences from different organisms: Leishmania mexicana,
Trypanosoma brucei (Tryp; SwissProt code: P13377), human [28], rabbit [25] and pig [27]. The secondary elements of N-terminally deleted glucose-6phosphate isomerase from Leishmania mexicana (PGI-Lm) are represented as h (a-helices) and s (b-sheet strands). The small-domain helix and sheet
elements are underlined. Residues in bold are involved in the coordination of the substrate’s phosphate group. Positions of the possible catalytic
residues, described in previous work [21], are colored in magenta. In the Leishmania (Leish) sequence, the residues shown in italics are not seen in the
electronic density maps of PGI-Lm; the PGI-Lm residues in the black box have a mean Ca r.m.s. deviation of 3.3 Å when superimposed to the
human PGI [28]. The PGI-Lm residues in the gray box superimpose with human PGI with a mean Ca r.m.s. of 0.9 Å. Loops A and B are colored
green and yellow, respectively. PGI-Lm residues, marked above the alignment using the letter ÔcÕ, are in contact with residues of the opposite
monomer by a distance of less than 3.6 Å. The ÔoÕ marks residues of PGI-Lm that are in hydrophobic contact with Met337 (marked with #).
human homologue [28] (pdb code, 1jlh), although both
present the same overall fold. The small domain from all
PGIs is connected to the large domain by two long a-helices
named LH-n (long helix connected to the small domain Nterminus) and LH-c (long helix connected to the small
domain C-terminus). The PGI-Lm small domain encompasses residues 197–316. The superposition of Leishmania
and human PGI (pdb code, 1jlh), using the large domain
and the first a-helix (h-l) of the small domain (Fig. 2), results
in a Ca-r.m.s. of 0.9 Å for the considered residues. The
calculated Ca-r.m.s. for the remaining residues (small
domain, except for the h-l a-helix) is 3.3 Å (Fig. 3B).
Superposition of just the small-domain residues, including
the h-l a-helix, from each monomer results in a mean Car.m.s. of 0.7 Å. This r.m.s. analysis indicates a rigid body
displacement between the large and small domains when
comparing human and Leishmania PGI.
The fact that dimer assembly is driven by contacts
between residues located exclusively in the large domain
(Fig. 1) suggests that residues connecting large and small
domains from the same monomer are responsible for the
differences between the PGI structures. Moreover, the high
Ó FEBS 2004
structural similarity of the small domain region (r.m.s. of
0.7 Å), observed between the mammalian and parasite
PGIs, point to a conserved packing of the secondary
structure elements of this domain. Amino acid substitutions
in the small domain do not result in significant alterations of
the domain interfaces between the two PGIs. We identified,
from this superposition analysis, that the presence of
Met337 in PGI-Lm, and Ala284 at a structurally equivalent
site in the human PGI, contribute significantly to the smalldomain position differences observed between both structures. Met337 is located in an a-helical segment connecting
the C-terminal side of the small domain to the LH-c of the
large domain (Fig. 2).
The first a-helix of the small domain (h-l) is positioned
adjacent to the large domain, establishing the main interdomain contacts (Fig. 3A). The mean Ca-r.m.s. for h-l,
calculated between human and PGI-Lm, is equivalent to the
Ca-r.m.s. calculated between the large domains (< 0.9 Å).
The direction of the h-l a-helix can be associated with an
imaginary axis describing a rigid body movement of the
small domain between the superimposed structures
(Fig. 3B). The substitution of Ala284 in human PGI with
Met337 in PGI-Lm causes a local stress in both long ahelices (LH-n and LH-c) connecting the large and small
domains. Met337 is located at a short a-helix segment (Hmet), side-by-side with h-l of the small domain, and makes
hydrophobic contacts to conserved residues in LH-c and
LH-n. The Ca atoms of Met337 in PGI-Lm and Ala284 in
human PGI are separated by a distance of 1.6 Å, while the
Ca atoms of the first residue of LH-n, Tyr342 (in PGI-Lm)
and Phe289 (in human PGI) are at a distance of 2.5 Å
(Fig. 3C). Finally, it is clear from the structural superposition that PGI-Lm presents its small domain in a more
open conformation when compared to the human homologue, resulting in a larger active-site cavity. This may
explain the difference in the affinity of the proteins for their
substrate and product. A higher Km value for the substrate
D-Fru6P was measured for PGI-Lm (242 lM) when compared with that of the human enzyme (99 lM) [14].
Substrate binding
The position of D-Fru6P in the dPGI-Lm structure is clearly
seen in the difference map calculated in the absence of the
ligand (Fig. 4A). However, as it is not clear whether
D-7Fru6P is in the open or closed conformation, both
Fig. 3. Structural details of the Leishmania PGI. (A) Cartoon representation of large (blue) and small (green) domains from Leishmania
mexicana glucose-6-phosphate isomerase (PGI-Lm). The large and
small domains are connected by two long a-helices, named LH-n and
LH-c; Met337 is located at a-helix ÔH-metÕ in the large domain, which is
placed side-by-side with Ôh-lÕ in the small domain (B). Superimposed
monomers from human (gray) [28] and PGI-Lm (green) highlight the 2
Å side-displacement of their small domains. An imaginary rotation axis
can be placed close to h-l, indicated by the vertices of the drawn lines.
(C) Detailed view of Met337 neighboring residues located in both LH-n
and LH-c of PGI-Lm (green). The small domain from human PGI is
represented by the gray surface. The presence of Met337 at the corresponding position of Ala284 in human PGI causes the displacement
of Tyr342 (Phe286 in human PGI) located in the LH-c of PGI-Lm.
Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2769
2770 A. T. Cordeiro et al. (Eur. J. Biochem. 271)
Ó FEBS 2004
Fig. 4. Stereoview of the active site of N-terminally deleted glucose-6-phosphate isomerase from Leishmania mexicana (dPGI-Lm) with bound
D-fructose-6-phosphate (D-Fru6P). (A) Electron density map 2rA|Fo|-D|Fc| (contoured at 1r), colored green, yellow and magenta for loop A, loop B
and D-Fru6P, respectively; monomers A and B are represented by gray and blue sticks, respectively. (B) Hydrogen bonds between D-Fru6P and
dPGI-Lm residues are colored yellow, while red dot lines represent the separation between atoms that form hydrogen bonds in the D-Fru6P/rabbit
PGI complex [21]. These distances indicate that loops A and B from PGI-Lm should move 5 Å to place D-Fru6P in a similar orientation
described for D-Fru6P/rabbit PGI.
possibilities were explored and finally a closed conformation
was chosen owing to a better fit with the difference map.
The comparison with the rabbit PGI/D-Fru6P [21] revealed
that the phosphate group of D-Fru6P interacts with the
same residues in both structures. The only exception is
Ser209 (Ser159 in rabbit PGI) which, owing to the open
conformation of dPGI-Lm, is making a hydrogen bond to
O4 of D-Fru6P. The other residues – Ser259, Lys260,
Thr261, Thr264 and Thr267 (corresponding to Ser209,
Lys210, Thr211, Thr214 and Thr217 in rabbit PGI) –
maintain a similar hydrogen bond network to the three
oxygen atoms of the phosphate group (Fig. 4B). The main
difference observed in the D-Fru6P coordination between
the mammalian and Leishmania PGIs is a result of the open
conformation of the active site of the latter. In the rabbit
PGI in complex with D-Fru6P [21], residues His388(b) and
Glu357 are less than 3 Å from the oxygen atom and
hydroxyl groups of the substrate ring. Based on this
enzyme–substrate complex structure, it was proposed that
His388(b) participates in the substrate ring opening step,
and Glu357 acts as a proton exchanger in the isomerization
step [27]. In dPGI-Lm, His441(b) makes a hydrogen bond
to O2 of D-Fru6P, and Glu410 is 7.7 Å distant from the
closest hydroxyl group of the D-Fru6P ring (Fig. 4B). To
bring D-Fru6P to a similar orientation, as observed in the
rabbit PGI complex, would require a movement of 5 Å in
the direction of Glu410. Two possibilities of such dislocation were analyzed.
The first would require a 2.5 Å displacement of the small
domain followed by a 2.5 Å movement of loops A and B.
These loops in the rabbit [29] and human [28] PGIs
comprise residues 209–215 and 246–262, respectively (numbered according to the human PGI sequence). Loops A and
B are known to undergo a rigid body displacement of
Ó FEBS 2004
Leishmania PGI crystal structure (Eur. J. Biochem. 271) 2771
Fig. 5. Neighboring PGI molecules contact surface. (A) Ribbon representation of deleted glucose-6-phosphate isomerase from Leishmania
mexicana (dPGI-Lm), showing the surface of a neighboring molecule
present in the crystal packing. Loop A, loop B and D-fructose-6phosphate (D-Fru6P), are colored green, yellow and magenta,
respectively. Each monomer forming the dimer unit is colored in gray
or blue. (B) Detailed view of hydrogen bond interactions in the crystal
interface and hydrophobic interaction between Phe260 (in loop A) and
Phe308 (in loop B). The close packing of dPGI-Lm symmetry-related
molecules restricts the movement of loops A and B. The flexibility of
these loops is necessary for the effective approximation of substrate to
catalytic residues His441 and Glu410 in the active site. The colored
patches in the symmetry-related molecule surface are related to the
presence of oxygen (red) or nitrogen (blue) exposed atoms.
2.5 Å in order to coordinate the phosphate group of the
substrate. Loops A (residues 256–262) and B (residues 298–
314) of PGI-Lm (Fig. 2) are structurally conserved and
capable of undergoing a similar displacement, as observed
for the mammalians PGIs. The movement, of 2.5 Å, of the
small domain, however, would result in improper contacts
with the surrounding residues, specifically with the Met337
described above.
Another explanation for making the required contact
would involve the movement of loops A and B by the whole
distance of 5 Å that separates the substrate from the
correct orientation with the catalytic residues (His441b and
Glu410a, according to the PGI-Lm sequence). The simultaneous movement of these loops is mediated by Phe260
located in loop A that makes contact with loop B, forcing it
to dislocate together with loop A. Residues of loop B are
accessible to solvent and would not be restricted in such
dislocation by interactions with other parts of the small
domain. The residues from loop B which are not accessible
to solvent – Val304, Phe307 and Ile309 – are in hydrophobic
contact with Phe261 of loop A. Such orchestrated movement of both loops would bring D-Fru6P closer to the
proposed catalytic residues, allowing the isomerization
reaction.
Crystal contacts
One area of crystal contact of symmetry-related dPGI-Lm
molecules occurs between Gln263 (loop A) and Lys304
(loop B) of one molecule and residues His80, Gln81 and
Gln85 of its symmetry-related molecule (Fig. 5). These
contacts represent the main hydrogen bonds between the
related molecules and, together with weaker interactions,
are believed to hold loops A and B in the open conformation observed in the crystal packing. These interactions have
allowed D-Fru6P to reach the correct phosphate coordination residues (Fig. 4A), but blocked the movement of the
flexible loops, obstructing the enzyme to close the catalytic
cavity, as seen in the rabbit-PGI/D-Fru6P complex [21]. The
crystalline contacts do not contribute to the orientation of
the small domain. The large and small domains from
Leishmania PGI form the unique and rigid body that is
different from the mammalians PGIs. The conserved loops
A and B are believed to be the only flexible region in PGI
structures.
Conclusions
Both a full-length and an N-terminally deleted form of
L. mexicana PGI were crystallized, the latter in the presence
of D-Fru6P, and their structures refined to 2.6 Å and
2.35 Å, respectively. A comparison of these structures with
those of human and other mammalian PGIs highlights
unique features of the parasite enzyme. Although the overall
fold of human and L. mexicana PGI appeared to be similar,
and the substrate-binding residues to be conserved, a
significant difference was detected in the position of the
2772 A. T. Cordeiro et al. (Eur. J. Biochem. 271)
small domain that presents a more open conformation in
the enzyme of the parasite. As a result, a larger active-site
cavity is created, providing a possible explanation for the
different values of kinetic parameters, as measured for the
human and parasite enzymes. This larger cavity may be
used in future rational drug design strategies, to develop
selective inhibitors of the parasite enzyme which are too
large to be accommodated by the catalytic site of the
mammalian PGI.
Acknowledgements
This work was supported, in part, by research grants 98/14979-7
(FAPESP) and 478127/01-4 (CNPq) to O. H. Thiemann. A. T.
Cordeiro is a recipient of a FAPESP student fellowship (project number
00/14960-6). We would like to thank the members of the Protein
Crystallography and Structural Biology Group (IFSC-USP) for helpful
discussions in the course of this work. We thank, in particular, Drs
Robert Sweet, Denize Kranz and Michael Becker, and all RapiData
2003 course staff for their assistance in data collection. Data for this
study were measured at beamline X25 of the National Synchrotron
Light Source, whose financial support comes principally from the
National Center for Research Resources of the National Institutes of
Health (NIH), and from the Offices of Biological and Environmental
Research and of Basic Energy Sciences of the US Department of
Energy.
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