A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics
Introduction
The so-called parasitic diseases affect today a great part of the world population, causing many deaths and having a great limiting influence in the life quality and development of several countries, especially in the tropics. These diseases are usually caused by complex organisms, mainly worms and protozoa.
Protozoa are responsible for most parasitic infections affecting human beings. Protozoonoses are caused by about 10.000 known species of protozoa and are most common at the less developed regions of the planet, being usually endemic. The most lethal protozoa disease in humans is malaria [1], which affects about 300 and 500 million people worldwide, causing between 1.0 and 2.5 million deaths annually, mostly among children. Today, the threat is more significant for nations in Africa, Asia, Latin America and some regions of the South Pacific. It is estimated that approximately 40% of the world population is at risk of infection with malaria [2]. This disease is caused by protozoa of the genus Plasmodium. In humans, four species are responsible for Malaria: Plasmodium falciparum (the most dangerous), P. vivax, P ovale and P. malariae.
Two aspects have currently stimulated new efforts regarding the development of chemotherapy, vaccines and sanitary studies about malaria: the rapid emergence of P. falciparum strains which are resistant to currently available antimalarial drugs [3], [4], [5], [6] and the inefficacy of antimalarial vaccines [7]. Since a vaccine for malaria seems to be far in the future, a more immediate solution would be the development of new antimalarial chemotherapy. Still, we have to assume that the ability of Plasmodium to adapt to new chemotherapy under the pressure of the drugs would eventually render any new antimalarial drugs less efficient, thus making all research on aspects of malaria even more important. Many strategies have been adopted to develop new drugs for malaria. Among them, we have been interested on the design of new inhibitors for three enzymes involved in the parasite methylenetetrahydrofolate cycle using molecular modeling and dynamics studies. It is well known that inhibitors of folate metabolism are quite important drugs, not only in the chemotherapy of malaria, but also of bacterial infections and cancer [8]. The effectiveness of antifolates is based on the perturbations they cause in the folate pathways, which rapidly lead to nucleotide imbalances and cell death [9], [10]. This turns the enzymes involved in this cycle into good targets to antimalarial chemotherapy. These three enzymes are thymidylate synthase (TS), dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase (SHMT). Among those enzymes, DHFR have been the main target for antimalarial chemotherapy mainly because this enzyme shows important active site differences for different species. For example, the similarity between the human and the P. falciparum DHFR active sites is only 45%, a fact that allows for the development of antifolates, which are very selective towards the parasite enzyme. We believe that this variability on the primary sequence of DHFR for different species is a clear indication of the potential of this enzyme to suffer important mutations in its active site without loss of its activity. In fact, those mutations are responsible for the widespread resistance of P. falciparum to antifolates.
The fact that all protozoa, including Plasmodium, have the enzymes DHFR and TS as parts of a bifunctional homodimeric enzyme, in contrast with mammalians that have these enzymes as monofunctional separate proteins, also opens a new perspective that could be used in the development of new and more selective antimalarials: targeting the mechanism of substrate transport from one active site to the other. We have already considered this possibility in a previous work [11].
The other enzyme of the folate cycle, SHMT, is a member of the α-class of the pyridoxal-5′-phosphate-dependent enzymes; it reversibly catalyses the conversion of serine into glycine, while the hydroxymethyl group is transferred to 5,6,7,8-tetrahydrofolate, transforming it in 5,10 methylenetetrahydrofolate, the sole precursor of purine biosynthesis and a key intermediate in the biosynthesis of thymidine, choline and methionine [12], [13]. This enzyme also catalyses THF-independent aldolytic cleavege, decarboxylation, racemization and transamination reactions [14], being ubiquitous in nature.
The indispensable role of SHMT in DNA biosynthesis, allied to the fact that a high level of this enzyme activity was observed in rapidly proliferating cells during the S phase of the cell cycle and in a wide variety of tumors cells [15], [16], [17], points to SHMT as a potential target for the development of anticancer and antimicrobial agents [15], [18], [19]. However, SHMT shows great sequence similarity between species. In fact, the similarity between the active site of the P. falciparum SHMT and the human enzyme is 86.95%. This fact makes the discovery of selective inhibitors for this enzyme quite difficult. On the other hand, if those inhibitors are somehow developed, it is likely that the natural selection for resistant strains would be much slower. Still, this enzyme has not been validated yet as a new target for antimalarial chemotherapy. While DHFRTS have been well studied in protozoan parasites [11], [20], [21], [22], [23], [24], little is known about parasitic SHMT [25], [26], [27], [28]. Its crystallographic structure is yet unknown, and only recently its gene organization was revealed [29].
In this paper, we propose a 3D model for P. falciparum serine hydroxymethyltransferase (pfSHMT) in complex with N-glycine-[3-hydroxy-2-methyl-5-phosphonooxymethyl-pyridin-4-yl-methane] (PLG) and 5-formyl tetrahydrofolate (5-FTHF) or 5-formyl-6-hydrofolic acid (FFO). This model was built based on homology modeling by multiple alignment using as templates the crystallographic structures of SHMT available in the Protein Data Bank (PDB) [30], [31].
Section snippets
Experimental
In the search for good templates, all the amino acid sequences of SHMT available in the PDB were used. The SHMTs from E. coli (eSHMT; PDB entry 1DF0, resolution=2,40 Aˇ, R-value=0,174) [32], Human (hSHMT; PDB entry 1BJ4, resolution=2,65 Aˇ, R-value=0210) [33], Murine (mSHMT; PDB entry 1EJI, resolution=2,90 Aˇ, R-value=0,271) [34], Rabbit (rSHMT; PDB entry 1CJ0, resolution=2,80 Aˇ, R-value=0,214) [35] and Bacillus stearothermophyllus (bsSHMT; PDB entry 1KKJ, resolution=1,93 R-value=0,178) [36]
Homology modeling
Fig. 1 shows our final alignment, which was submitted to the SWISS Model server to build the initial model of pfSHMT. This alignment was obtained after manual adjustments of the initial alignment from the BLAST server [37], [38]. The adjustments were performed after inspection of the alignment of the pfSHMT sequence with the sequences of other species proposed by Alfadhli and Rathod [29] and the alignment between the sequences of eSHMT, rSHMT, mSHMT and hSHMT proposed by Scarsdale et al. [35].
Conclusions
In this work, we have used homology modeling and molecular dynamics studies to propose the first 3D structure for pfSHMT. The comparison between the known crystallographic structures for several SHMT and our pfSHMT model indicates that the Plasmodium enzyme could be a dimer in solution. We have also used molecular dynamics and docking studies to explore the opportunities opened by the differences found for the interactions of a monoglutamate tailed substrate with the active site of the pfSHMT
Acknowledgments
The authors wish to thank the Brazilian financial agencies CNPq, FAPERJ and CAPES for financial support and Dr. Alan Wilter Souza da Silva for his help with the GROMACS Package.
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