HOMOLOGY MODELLING AND BINDING
SITE IDENTIFICATION OF 1 DEOXY
D - X Y L U L O S E 5 P H O S P H A T E
REDUCTOISOMERASE OF PLASMODIUM
FALCIPARUM: NEW DRUG TARGET
FOR PLSMODIUM FALCIPARUM
JYOTSNA CHOUBEY* 1, ASHISH PATEL1, SHAILENDRA GUPTA2, M.K.VERMA1
1
Sub-DIC Bioinformatics,National Institute of Technology Raipur(C.G.) India
2
Indian Institute of 4062385 Toxicology Research Lucknow(U.P.) India
Abstract : Malaria is major global health problem. Malaria parasite had developed resistance to the drug being used till date. It implies the development of new effective drug with different mode of action. Apicoplast in malaria and related parasite offer various new target for drug therapy[1]. Apicoplast contains various metabolic pathways that differ from those of host thereby presenting ideal strategies for drug therapy. Plasmodium falciparum 1deoxy- D- xylulose 5- phosphate reductoisomerase (pfDXR) is a potential target for antimalarial chemotherapy. The three dimentional model (3D) of this enzyme was determined by means of homology modeling through multiple alignment followed by intensive optimization and validation. The comparative modeling of pfDXPR was performed by using comparative modeling program MODELLER, Swiss Model, 3Djigsaw, and Geno3D.The modelling of the three dimensional structure of pfDXPR shows that models generated by Modeller were more acceptable in comparison to that by 3Djigsaw, Geno3D and Swiss Model. The obtained models were verified with the structure validation programs like, PROCHECK & Swiss pdb viewer was used for energy refinement of the model. Self Optimized Prediction Method with Alignment (SOPMA) is employed for calculating the secondary structural features of pfDXR protein sequences considered for this study. The secondary structure indicates whether a given amino acid lies in a helix, strand or coil. The results revealed that alpha helix dominated among secondary structure elements followed by random coils, extended strand and beta turns for all sequences. Active site determination through CASTp suggests that this protein can acts as potential drug target.
Keywords: Apicoplast, Chemotherapy, Homology modeling, pfDXPR, Active site determination
otherwise do not attack eukaryotes. However, most apicoplast proteins are encoded in the nucleus and then transported to the apicoplast by a specificmechanism involving two amino-terminal targeting sequences (Fothet al., 2003; Waller et al., 1998). Apicoplast biology includes a number of biochemical pathways that are present in bacteria, plants and apicomplexan parasitesbut are absent in the human host and thus provide obvious opportunities for chemotherapy (Ralph et al., 2001; Roos et al., 2002). Isopentenyl diphosphate, the precursor for isoprenoids, is synthesizedin plants and animals via the mevalonate pathway, but an alternative pathway,known as the 1-deoxy-D-xylulose 5-phosphate (DOXP) or non-mevalonatepathway, is present in bacteria and chloroplasts. Genes encoding two enzymes in this pathway, DOXP reductoisomerase and DOXPsynthase, are encoded by P. falciparum and contain putativeapicoplast targeting signals (Jomaa et al., 1999). The antibioticfosmidomycin inhibited the activity of recombinant DOXP reductoisomerase,inhibited the growth of cultured P. falciparum parasites andcured murine malaria (Jomaa et al., 1999). Fosmidomycin waspreviously developed as an antibacterial, so it could quite quickly be brought to human trials for malaria.
Materials and Methods: The sequence of 1 Deoxy-D-Xylulose-5-Phosphate Reductoisomerase of plasmodium falciparum( pfDXPR) was taken from NCBI database(ID code ABQ23790.1).Structure of pfDXPR has been based on the availability of the template sequence of 1 Deoxy-D-Xylulose-5-Phosphate Reductoisomerase of mycobacterium tuberculosis(mtDXPR pdb code2C82).The search for sequence similarity within database has been performed with BLAST program.The program MODELLER was used to build the model of pfDXPR according to the homology modeling method. The steriochemical quality of models was verified with the program PROCHECK(Laskowski et al.,1996 ) in order to select best model.The fasta sequence of pfDXPR was submitted to 3Djigsaw, Geno3D(Combet et al.,2002), and SWISS MODEL SERVER(Arnold et al., 2006). and the structure was obtained on email address. The energy minimization of all the structures modelled by different homology modeling methods was done with the hlp of offline tool SPDV(Guex and Manuel,1997). The structure modeled by MODELLER, GENO3D, 3DJIGSAW, and SWISS MODEL SERVER were having energies 13076.054, -16397.348, -4844.366, -6766.616 KJ/mol respectively.So the minimum energy of was found for structure modeled by MODELLER and GENO3D.The evaluation of the four generated model of pfDXPR was performed by two tools-PROCHECK and WHAT IF(Vriend,1990). The secondary structure evaluation was done with the help of SOPMA (Geourjon and Deleage,1995). Active site determination was done with the help of CASTp(Dundas et al, 2006) for the modeled protein to further work on its docking studies. Active site determination gives us an idea to make a grid before docking.
RESULT AND DISCUSSION: The modelled structure of pfDXPR(fig1) obtained by homology modeling contains alpha helix,beta sheets,and loops( Fig.1).
3djigsaw swiss model server
Modeller
Geno3D
The PROCHECK result obtained for pfDXPR by four different methods are as follows:
GENO3D SWISS MODEL
a) b)
3D JIGSAW MODELLER
c) d)
Fig.2: Ramchandran plot of the structure of pfDXPR obtained through various modeling tool
The structure modeled by GENO3D (fig.2(a)), SWISS MODEL (fig.2(b)), 3D-JIGSAW (fig.2(c)), MODELLER(fig.2(d)) shows the amino acid percentage in the favourable region as 87.4%,84.0%, 76.2%, and 88.0%respectively and amino acid percentage in disallowed region as 0.2%, 0.6%, 2.2%,0.6% respectively. This shows that model builded by modeler shows the highest percentage of allowed region amino acids. The next comparision was done by secondary structure evaluation with the help of Self Optimized Prediction Method with Alignment (SOPMA). The secondary structure indicates whether a given amino acid lies in a helix, strand or coil. Secondary structural features as predicted using SOPMA are represented in Table1. The results revealed that alpha helix dominated among secondary structure elements followed by random coils, extended strand and beta turns for all sequences. The secondary structure are predicted by using default parameters (Window width: 17, similarity threshold: 8 and number of states:4).
Secondary Structure
Alpha helix 42.73% 310 helix 0.00%
Pi helix 0.00% Beta bridge 0.00% Extended strand 17.55%
Beta turn 7.39%
Bend region 0.00% Random coil 32.33% Ambigous states 0.00% Other states 0.00%
CASTp: Computed Atlas of Surface Topography Of Proteins() gives prediction of actve site and the number of amino acid involved in it. The server gave the best active site as a area of 1655.8 and volume of 2519.9 amino acid are involved in the active site formation.(Fig.3)
Fig.3: Active site information by CASTp
The green colour (fig.3) shows the active site position of the builded protein starting from 44 amino acid to some interaction of 391 amino acid. The major coverage of amino acid to be in binding cavities lies in between 44-50,161-169,187-194 and251-278.
CONCLUSION: The alpha helices were obtained from position 48-432 with beta sheet in between them. A little variation was obtained between Modeller and Gen3D structure prediction.The energy value for both the structure and Ramachandran plot was also almost similar. So the best structure are modelled by MODELLER and GENO3D, then the active determination by CASTp gives us an opportunity to further work on its docking studies and its binding activities.
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