Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase
© Monzani et al; licensee BioMed Central Ltd. 2007
Received: 12 March 2007
Accepted: 25 September 2007
Published: 25 September 2007
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 188.8.131.52) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design.
The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP.
Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.
Most known organisms synthesize purine bases by two pathways. The de novo biosynthesis pathway builds the purine nucleotide on 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP). The salvage pathway recovers purines (adenine and guanine) from the degradation products of nucleotide metabolism and from hypoxanthine and xanthine. In contrast, parasitic protozoa such as the members of the Kinetoplastida order are auxotrophs for purine bases because the de novo biosynthetic pathway is completely absent . They are therefore dependent on recycling pre-formed purine nucleotides and acquiring purines from the host. Central to the salvage pathway are the phosphoribosyltransferases (PRTases). In Kinetoplastids in general and Leishmania in particular, three PRTases are involved in the recycling of purine bases by the salvage pathway: hypoxanthine-guanine PRTase (HGPRT) (EC 184.108.40.206), adenine PRTase (APRT) (EC 220.127.116.11) and xanthine PRTase (XPRT) (EC 18.104.22.168) . Several PRTases have been characterized from different organisms, but crystallization and structure determination have been accomplished for only two HGPRTs from Kinetoplastids, the parasite Trypanosoma cruzi  and L. tarentolae (present work).
PRTases are classified as Type I and Type II depending on their structural and catalytic features. The best-studied PRTases belong to the 'Type I' group, sharing a common α/β-fold at the PRPP binding motif and a flexible loop, besides a core region of at least five parallel β-strands surrounded by three or more helices [4, 5]. The 'Type II' PRTases are composed of a mixed α/β N-terminal domain and an α/β barrel-like C-terminal domain. Currently, Mycobacterium tuberculosis and Salmonella typhimurium quinolinate PRTases are the only known structures in this group [6, 7].
Considerable interest in the salvage pathway as a potential target for chemotherapy has been stimulated by the differences in purine base metabolism between mammalian hosts and protozoan parasites [2, 8]. The creation of independent Δhgprt, Δaprt and Δxprt null mutants by targeted gene replacement in L. donovani cells revealed that all three of the knockout strains generated are viable in the mouse macrophage model . However, the Δhgprt/Δxprt double mutant L. donovani strain has less than 5% of the wild-type capacity to infect macrophages, establishing HGPRT and XPRT as essential for purine acquisition, parasite viability and infectivity in the mouse model .
L. tarentolae has been exploited as a model Leishmania for a variety of molecular, biochemical and evolutionary studies because of the ease of cell culture and genetic analysis of this species. In this paper we describe the three-dimensional structure of a L. tarentolae HGPRT protein and compare it with other HGPRT structures deposited in the Protein Data Bank. In view of the close phylogenetic relationship, the results will be of general significance as a model for other species of pathogenic Leishmania.
Results and discussion
Crystallographic data summary
unit cell (Å)
a = 58.1, b = 85.4, c = 87.6
Matthews' volume (Å3/Da)
solvent content (%)
Data reduction statistics (a)
high resolution limit (Å)
observed unique reflections
Model refinement statistics
high resolution limit (Å)
number of protein atoms (2 monomers) (b)
number of solvent molecules
number of GMP atoms (2 molecules) (b)
average isotropic B-factor (protein – Å2)
bond lengths (Å)
bond angles (°)
torsion angles (°)
improper angles (°)
Ramachandran plot (%)
⟨real-space CC⟩ (c)
No. Of residues with real-space CC < (⟨CC⟩ – σ CC )
Directional atomic contact analysis (d)
all contacts Z-score
backbone-backbone contacts Z-score
backbone-side chain contacts Z-score
side chain-backbone contacts Z-score
side chain-side chain contacts Z-score
The monomer structure
The core domain consists of a central five-stranded parallel β-sheet (strands β3, β2, β4, β5 and β6), with one α-helix packed on each side of the sheet (helices α2 and α3). A small 310 – helix (η2) is present in the core domain. The central β-sheet is formed by two β/α/β motifs joined side-by-side through the first strand of each motif (β2 and β4). One further strand (β6) completes the central β-sheet. A phosphate binding site is present in the loops between the first β-strand and the α-helix of the β/α/β motif (called loops I and III respectively). Loops I and III are involved in binding the two terminal phosphates of PRPP [11–13]. L. tarentolae HGPRT Loop III residues Asp129, Ser130, Ala131 and Thr133 interact with the GMP phosphate group.
A flexible loop (loop II, residues 92–118), the function of which may be related to the formation of the transition state [11, 14, 15], comprises a region (residues 106–114) with good stereochemical and statistical values, which adopts different conformations in the two chains. In chain B, the polypeptide partially forms an α-helix (αL) similar to that in human HGPRT , while in chain A this helix is not observed and the polypeptide conformation resembles that found in T. cruzi HGPRT , Tritrichomonas foetus HGXPRT  and E. coli HPRT . The different conformations adopted by these residues in the two monomers are consistent with the flexibility generally observed in the equivalent region (92–118) of other HGPRTases.
The hood domain contains both the N- and C-termini and is constituted by a small anti-parallel β-sheet with three strands (β1, β7 and β8). A loop (loop IV, residues 175–195) connects the β-strand β6 in the core domain to β8 in the hood-domain. This loop (IV) contains some of the residues that bind the base of GMP by hydrogen bonding with Val179 and Asp185 and by hydrophobic interaction with Phe178. The other connection between the core and the hood domain is made by a long α-helix (α1), which ends with a small 310 – helix (η1). This helix appears to be important for the structural stability of HGPRT, since it interacts with all strands of the central β-sheet and with helix α2 of the core domain and is also involved in dimerization contacts.
The dimeric interface
Structural water molecules in each monomer (H2O1 and H2O18) stabilize the polar side chains of Thr37 and Tyr182 by hydrogen bonds that are found in a hydrophobic region formed by the Trp34, Val33, Phe71 and Phe78 side chains. Moreover, Thr37 and Tyr182 in both Leishmania and Human HGPRT form hydrogen bonds to neighboring Val33 and Asp74, respectively. This water molecule stabilization is exclusively observed in the Leishmania structure; in homologous structures, Thr37 is substituted by a hydrophobic residue.
Comparison of HGPRT structures
The known HGPRT structures of E. coli, S. typhimurium, Thermoanaerobacter tengcongensis, T. foetus, T. cruzi, P. falciparum, Toxoplasma gondii and the human enzyme were compared with L. tarentolae HGPRT. The sequences were aligned (not shown) and the structures superposed.
A non-proline cis peptide bond between Leu65 and Lys66 from loop I is conserved in type I PRTases [3, 5, 11, 16, 17, 21], where the amide nitrogen of Lys66 is exposed to the active site so that the peptide bond contributes two adjacent hydrogen bonds to the PRPP-metal complex . However, our structural comparison of HGPRTs (Figure 3) suggests that the Lys66 cis conformation acts in the communication between monomers and drives the Arg191 side chain toward the active site into the correct position to bind PPi (Figure 3). Structures with a cis conformation in complex with PRPP as well as with PPi give strong evidence for this [11–14, 22, 23].
Interaction distances between active site residues and GMP
Residue L HGPRTc
Leishmania HGPRT inhibition tests
IC50 values of purine and pyrimidine analogs for Leishmania HGPRT
HGPRT is a known activator of purine base analogs such as 6-mercaptopurine and allopurinol, and has been proposed as a target for antiparasitic chemotherapy [2, 8]. Allopurinol is metabolized by HGPRT and incorporated into RNA during transcription, resulting in its degradation and inhibition of protein synthesis . Allopurinol is metabolized more efficiently by the parasite HGPRT than the human homologue [25–27], prompting its evaluation in the treatment of leishmaniasis  and Chagas disease [25, 29] with promising results.
The X-ray structure of L. tarentolae HGPRT with GMP bound at the active site provides the template for comparison with the human enzyme. The subtle differences observed between the parasite and the human enzyme in the contacts with ligand can be explored for the design of potential parasite-specific inhibitors. The dimeric structure of L. tarentolae HGPRT shows an intricate hydrogen bond network important for enzyme stability and required for its activity. This analysis, together with the inhibition experiments using purine and pyrimidine analogs, has revealed differences in the binding efficiency of the enzyme active site that could be explored in the development of further inhibitors.
Protein expression, purification and crystallization
The recombinant HGPRT of L. tarentolae was over-expressed in E. coli BL21(DE3), and purified and co-crystallized with GMP in 19% PEG 4000, 20.6% isopropanol, 5% glycerol, 95 mM tri-sodium citrate, pH 5.6, as described previously . Crystals grown under those conditions belonged to the primitive orthorhombic space group P 212121 (a = 58.1Å, b = 85.4Å, c = 87.6Å, α = β = γ = 90°). Crystals of the free enzyme, which were also obtained at 18°C in 15% PEG 6000, 100 mM citrate, pH 5.1, diffracted poorly, and the use of additives led to crystals that diffracted only up to 3.0Å. Co-crystallization with GMP led to better-diffracting crystals up to a resolution of 2.1Å and these were used to solve the atomic structure of Leishmania HGPRT.
Data collection and processing
L. tarentolae HGPRT crystals were transferred to a cryoprotectant solution without GMP, obtained by diluting the crystallization reservoir solution with 15% ethylene glycol (final concentration), mounted on nylon loops, and flash-cooled to 100 K. Diffraction data were collected at the Brazilian Synchrotron Light Laboratory with monochromatic X-rays (λ = 1.537Å) and a MAR345 image plate as detector . Two sets of consecutive diffraction images (75 and 62 images respectively, with 1° rotation per image) from the same single crystal were collected and processed. The diffraction images were indexed and integrated using DENZO . SCALEPACK  was used to scale and merge the data up to 2.1Å resolution. The data reduction statistics are summarized in Table 1.
Structure solution and refinement
The crystal structure was solved by molecular replacement using the deposited structure of the dimeric HPRT of T. cruzi  as search probe (PDB entry 1TC1; 55% sequence identity). X-ray data in the 20–2.3Å resolution range were used and one dimeric probe was positioned in the asymmetric unit during the molecular replacement procedure (program AMoRe) . The molecular replacement solution had a correlation coefficient of 57% and an R-factor of 43.5%. The molecular replacement model was refined iteratively in reciprocal and in real space using automated procedures and visual manipulation. Reciprocal space refinement was initially performed using the torsional simulated annealing procedure implemented in the CNS program  and continued using REFMAC5  from the CCP4 suite (Collaborative Computational Project, Number 4, 1994), using a maximum-likelihood target with stereochemical restraints, two TLS  sets of parameters (one for each protein monomer in the asymmetric unit), and individually restrained isotropic B-factors. A set of structure factors representing 5% of the total experimental data was excluded from the reciprocal-space refinement target for purposes of cross-validation. Twofold non-crystallographic symmetry restraints were used initially and gradually removed during the refinement. The program O  was used to inspect the (D|F o |-m|F c |) and (2D|F o |-m|F c |) difference Fourier maps and to manipulate the model. Water molecules were added automatically to the model on the basis of the difference Fourier maps and distance criteria using the program ARP/wARP version 5.0  from the CCP4 suite.
The stereochemical quality of the crystallographic model was constantly monitored during refinement using the PROCHECK , WHAT IF  and O  programs. The model/experimental map correlation was calculated using the MAPMAN© program . The refined TLS parameters and the residual isotropic atomic B-values were converted to atomic anisotropic displacement parameters using the program TLSANL  from the CCP4 suite.
The HGPRT enzyme inhibition assay was performed for 1 min in a 1 ml reaction volume containing 100 mM Tris-HCl, 5 mM MgSO4, 1 mM PRPP, 0.04 mM guanine at pH 7.4 . An extinction coefficient of 4.2 was used. Purine and pyrimidine analogues were tested using six inhibitor concentrations, in triplicate, to obtain the inhibition curve and calculate the IC50 values shown in Table 3.
This research was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP. The authors would like to thank Humberto D'Muniz Pereira by the assist the diffraction X-ray collection and the anonymous reviewers for their constructive comments.
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