Original Article

A computational approach to explore Plasmodium falciparum 3D7 Chorismate Synthase

N Arora, U Chari, A Banerjee, U Murty

Keywords

chorismate synthase, malaria, shikimate pathway

Citation

N Arora, U Chari, A Banerjee, U Murty. A computational approach to explore Plasmodium falciparum 3D7 Chorismate Synthase. The Internet Journal of Genomics and Proteomics. 2006 Volume 3 Number 1.

Abstract

Malaria remains the leading cause of deaths attributable to a communicable disease globally, with an unprecedented death toll of over 2 million per year. The reemergence of drug-resistant Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on Shikimate pathway, as it may provide the urgently required novel drug targets owing to lack of homologs of enzymes involved in the this pathway in humans. Chorismate Synthase, the key enzyme of Shikimate pathway, catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate to Chorismate via a 1, 4-trans elimination of phosphate. The strict requirement for a reduced flavin mononucleotide and the anti 1, 4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. The protein sequence of Chorismate Synthase containing 527 residues was obtained from NCBI. The enzyme was characterized using in silico tools. The secondary and tertiary (3D) structure of the enzyme was predicted using CS from other species and validated by various structural quality validation tools. Results of the current study will provide a deep insight about the structure and function of Chorismate Synthase and aid in rational drug designing.

 

Introduction

Each year, up to three million deaths due to malaria and close to five billion episodes of clinical illness possibly meriting anti-malarial therapy occur throughout the world, with Africa having more than 90% of this burden(Joel et al., 2004). Malaria was ranked the 8th highest contributor to the Disability Adjusted Life Year (DALY) causing almost 3% of DALY globally (WHO, 2002). This pandemic affects poorest of population residing in 107 countries (Hay et al., 2004, World Malaria Report, 2005). The ordeal due to morbidity and debility and mammoth loss of productive man hours is immense. The resurgence of drug-resistant apicomplexa, in particular P. falciparum, the most fatal human malarial parasite, has focused attention on the recent discovery of the Shikimate pathway in these organisms, as it may provide the urgently required, novel drug targets resulting from the absence of this pathway in mammals (McConkey et al., 2004). In addition, the pathway in apicomplexa is important for the supply of folates (Roberts et al., 1998, Roberts et al., 2002), compounds for which animals rely exclusively on an exogenous source. Moreover, some of the enzymes of the pathway catalyse biochemically unique reactions in nature, making them excellent targets for new antiparasite drugs. However, in order to assess the potential of the pathway in providing novel targets, the individual enzymes themselves need to be characterized (Ridley, 2002). Chorismate Synthase, the last enzyme of the Shikimate pathway, is itself biochemically unique in nature and catalyses a 1, 4-elimination of the 3-phosphate group and the (6R)-hydrogen from EPSP. In addition, the enzyme has an absolute requirement for reduced flavin mononucleotide (FMN) even though there is no net redox change in the overall reaction. As the catalytic action of Chorismate Synthase is biochemically unique in nature, the information supplied here is expected to begin to pave the way for developing an effective and specific antiparasitic agent. It has been inferred that there is not much information available on characterization of CS protein sequence of P. falciparum 3D7 involved in Chorismate synthesis using bioinformatics tools. Hence the focus of present work is to characterize CS protein (XP_966212.1) of P. falciparum 3D7 by using online and off-line bioinformatics tools.

Methodology

Sequence analysis of Chorismate Synthase (CS) of

P. falciparum 3D7 was selected as the candidate organism for the present study whose complete genome sequence is available. The protein sequence of P. falciparum 3D7 Chorismate Synthase (gi | XP_966212.1) was collected from NCBI protein sequence database (www.ncbi.nlm.nih.gov). Similarity search was performed using Position Specific Iterative (PSI)-BLAST against non-redundant database keeping default parameters like E- value threshold 10, word size 3 and Blosum 62 Matrix. Total 3 iterations of PSI-BLAST were considered as the BLAST search results converged after 3 iterations (Table 1). Since the BLAST algorithm detects local as well as global alignments, regions of similarity embedded in otherwise unrelated proteins can be detected (Altschul, 1997).The derived homologues sequences of CS protein were collected (Table 2) and best 9 BLAST hits were selected for further analysis. Multiple sequence alignment was performed using CLUSTALW (Higgins et al., 1994). Regions of conservation and variation were detected from CLUSTALW result (Fig. 1).

Phylogenetic analysis

A phylogenetic tree was constructed with the help of PHYLO_WIN tool (Galtier et al., 1998) Both Neighbor Joining and Maximum Likelihood methods were tried and the consensus tree was obtained (Fig. 2). Further Principal Component Analysis was performed to cross check the vicinity of the organisms based on the sequence information. (Fig. 3)

Physico-chemical Characterization of target Chorismate Synthase (CS): The basic physico-chemical properties of the CS protein sequence were calculated using the ProtParam tool (http://expasy.org/tools/protparam.html). The parameters computed by ProtParam are molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Kyte and Doolittle, 1982) (GRAVY). The GRAVY value for a peptide or protein is calculated as the sum of hydropathy values of all the amino acids divided by the number of residues in the sequence. All the results are tabulated in Table 3, 4 and 5.

Calculation of extinction coefficient: The extinction coefficient indicates how much light a protein absorbs at a certain wavelength. An estimation of this coefficient is useful for protein purification. Estimation of the molar extinction coefficient of a protein from knowledge of its amino acid composition is possible (Gill and Hippel, 1989). From the molar extinction coefficient of tyrosine, tryptophan and cystine (cysteine does not absorb appreciably at wavelengths >260 nm, while cystine does) at a given wavelength, the extinction coefficient of the native protein in water can be computed using the following equation (Gill and Hippel, 1989):

E (Prot) = Numb (Tyr)*Ext (Tyr) + Numb (Trp)*Ext (Trp) + Numb (Cystine)*Ext (Cystine)

Where (for proteins in water measured at 280 nm): Ext (Tyr) = 1490, Ext (Trp) = 5500, Ext (Cystine) = 125. The absorbance (optical density) can be calculated using the following formula: Absorb (Prot) = E (Prot) / Molecular weight.

Calculation of Instability index (II): The instability index provides an estimate of the stability of protein in a test tube. Statistical analysis of 12 unstable and 32 stable proteins has revealed (Guruprasad et al., 1990) that there are certain dipeptides, the occurence of which is significantly different in the unstable proteins compared with those in the stable ones. This method assigns a weight value of instability to each of the 400 different dipeptides (DIWV). Using these weight values it is possible to compute an instability index (II) which is defined as:

Figure 1

Where: L is the length of sequence

DIWV(xx [i+1]) is the instability weight value for the dipeptide starting in position i. A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable.

Calculation of Aliphatic index: The aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine) (Ikai, 1980). It may be regarded as a positive factor for the increase of thermostability of globular proteins. The aliphatic index of a protein is calculated according to the following formula (Kyte and Doolittle, 1982):

Aliphatic index = X (Ala) + a * X (Val) + b * (X (Ile) + X (Leu))

Where X (Ala), X (Val), X (Ile), and X (Leu) are mole percent (100 X mole fraction) of alanine, valine, isoleucine, and leucine.

The coefficients a and b are the relative volume of valine side chain (a = 2.9) and of Leu/Ile side chains (b = 3.9) to the side chain of alanine.

Calculation of half-life of a protein: ProtParam relies on the “N-end rule”, which relates the half-life of a protein to the identity of its N-terminal residue; the prediction is given for 3 model organisms (human, yeast and E.coli). The N-end rule originated from the observations that the identity of the N-terminal residue of a protein plays an important role in determining its stability in vivo (Bachmair et al., 1986; Gonda et al., 1989; Tobias et al., 1991, Ciechanover et al., 1989, Varshavsky et al., 1997).

Functional Characterization of CS: Functional characterization of CS protein sequence was done by finding motif using Eukaryotic Linear Motif (ELM tool (http://elm.eu.org) (Puntervol et al., 2003) and domain analysis was carried out using PROTSCAN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html).

Structure prediction of P. falciparum 3D7 Chorismate Synthase:

Secondary structure prediction: SOPMA tool (Geourjon and Deleage, 1995)( http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) was used to obtain the secondary structure of CS protein (Table 8 and Fig.5). The secondary structure prediction is the definition of each residue into either alpha helix, beta sheet or random coil secondary structures.

3D structure prediction: A comparative 3D structure analysis of P. falciparum 3D7 CS protein was performed using HOMER (http://protein.cribi.unipd.it/homer/ ) (Tosatto, 2005) and other web based servers (Table 9 and 10).

Evaluation of the obtained models of CS protein: The result was evaluated using PROCHECK(http://swissmodel.expasy.org/workspace/index.php?func=tools_structureassessment1&userid=USERID&token=TOKEN).The PROCHECK suite of programs assess the “stereo-chemical quality” of a given protein structure. The aim of PROCHECK is to assess how normal, or conversely how unusual, the geometry of the residues in a given protein structure is, as compared with stereo-chemical parameters derived from well-refined, high-resolution structures. Results from other web based servers were compared.

Results

The amino acid sequence of CS protein was retrieved from NCBI and PSI-BLAST program was used to find out the sequences that shared structure and sequence similarity against non- redundant database (Table 1). The results suggest that protein sequence with XP_743671.1 which belongs to Plasmodium chabaudi showed the highest degree of similarity (64% identity) to CS protein as query sequence, indicated by E-value and score.

Figure 2
Table 1: PSI BLAST iteration number and number of Hits obtained

Figure 3
Table 2: Summary of PSI BLAST iteration 3 (www.ncbi.nlm.nih.gov/BLAST/) of CS protein sequence when searched against non-redundant database

Further, CS protein was subjected to CLUSTALW for multiple sequence alignment and phylogenetic analysis (Fig. 1). The result suggests that CS protein of P. falciparum 3D7 was very much similar to CS from Plasmodium chabaudi.

Figure 4
Figure 1: Multiple Sequence Alignment using CLUSTALW as visualized by Jalview.

A phylogenetic tree was constructed using Neigbour Joining method (NJ) with100 bootstrap and 500 bootstraps replicate by using PHYLO_WIN. (Fig. 2).

Figure 5
Figure 2: Unrooted tree with 100 bootstraps and 500 bootstraps replicate obtained using Neighbor Joining method.

Figure 6
Figure 3: Principal Component analysis result

Figure 7
Table 3: Amino acid composition of Chorismate Synthase of

Figure 8
Table 4: Atomic composition of Chorismate Synthase

Figure 9
Table 5: Physico-chemical properties of Chorismate Synthase of .

# Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.+ Abs 0.1% (=1 g/l) 0.821, assuming ALL Cys residues appear as half cystines.* Abs 0.1% (=1 g/l) 0.809, assuming NO Cys residues appear as half cystines.++ Estimated half-life: The N-terminal of the sequence considered is M (Met).The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro). >20 hours (yeast, in vivo) and >10 hours (Escherichia coli, in vivo).
~ Instability index: The instability index (II) is computed to be 42.90; this classifies the protein as unstable.

The function of CS protein of P. falciparum 3D7 was analyzed by submitting the amino acid sequence to Eukaryotic Linear motif (ELM) server (www.elm.eu.org). Based on EML and PROSCAN results, the following motif and domains were assigned to CS protein amino acid sequence (Table 6 and 7).

Figure 10
Table 6: Motif analysis of Chorismate Synthase

Figure 11
Figure 4: Schematic representation of different types of predicted regions

Figure 12
Table 7: Domains obtained in Chorismate Synthase Protein using PROSCAN

Secondary structure prediction:

Figure 13

Figure 16
Figure 5: Secondary structure prediction of CS protein of by SOPMA tool.

Figure 15
Table 8: Percentage and type of Secondary structures present in CS

Figure 17
Figure 6: 3D structure of Chorismate Synthase

SOPMA program that was used to predict secondary structures in P. falciparum 3D7 suggest that it contained more helices than beta sheets. Window width was kept at 17, similarity threshold: 8 and number of states: 4 were used as parameters for analysis.

Three dimensional structure of CS of P. falciparum 3D7 was predicted by HOMER (http://protein.cribi.unipd.it/homer/) using template of Chorismate Synthase of Saccharomyces cerevisae (PDBID 1R53) keeping default parameters.

Figure 18
Figure 7: Ramachandran plot of modeled protein obtained using PROCHECK

The model was stereo chemically evaluated using the program PROCHECK. Through the inspection of the Psi/Phi angles of a Ramachandran plot obtained from this analysis, the backbone conformation of the model was evaluated. The overall conformation of the backbone was in good agreement with the stereochemistry, which was also found to be reliable.

Figure 19
Table 9: Model quality of CS protein obtained from different servers based on Ramachandran Plot using PROCHECK

Other web based servers were also employed to generate the 3 dimensional structure of P. falciparum 3D7 Chorismate Synthase and the results were compared.

Figure 20
Table 10: Model quality of CS protein obtained from different servers based on bond lengths, bond angles and planar groups using PROCHECK

{image:20}

Based on these evaluations, we conclude that model obtained using HOMER is superior as compared to models generated using other web based servers.

Discussion

In 2004, 107 countries and territories were reported to show vulnerability to malaria transmission. These statistics indicates the severity of malaria as the pre-eminent tropical disease and it is rated as one of the top three killers among communicable diseases. Anti-malarial drug resistance is recognized to be one of the greatest coercion to our ability to battle against malaria. The situation continues to be more frightening, with the geographical spread of resistance widening to previously unaffected areas and a ruthless augmentation both in the incidence and degree of drug resistance.

Selection and validation of novel molecular targets have become of paramount importance in light of the plethora of new potential therapeutic drug targets that have emerged from genomics revolution where we visage an avalanche of data but only flakes of information. With the increasing drug resistance in Plasmodium, there is an imperative need for exploring novel drugs to reduce the impending impact of the emergence of multidrug-resistant P. falciparum. Shikimate biosynthetic pathway is critical and specific for P. falciparum and other apicomplexan parasites but absent in mammals. The description of metabolic pathways, particularly those differing from humans, provides new targets for drug development. Computational methods play a crucial role in accelerating the drug development process and among them; comparative protein modelling is of great assistance during the rational design of drug molecules. In the dearth of experimental data, model-building on the basis of the known three dimensional structure of a homologous protein is the only unfailing method to obtain structural information. Hence in the present study we have focused to characterize the CS protein of P. falciparum 3D7 from sequence and structure and in elucidating function using bioinformatics tools. This study will provide a new insight into the structure of P. falciparum 3D7 Chorismate Synthase for rational designing of inhibitors in our crusade against this disease.

Acknowledgement

Authors are thankful to Dr. J.S. Yadav, Director, Indian Institute of Chemical Technology for his continuous support and encouragement. Neelima Arora thanks CSIR for Senior Research Fellowship.

Correspondence to

U.S.N Murty Deputy Director/ Scientist “F” Head, Biology Division, Indian Institute of Chemical Technology, Hyderabad-500007, India. Email: murty_usn@yahoo.com Phone: +91 40 27193134; Fax: +91 40 27193227

References

r-0. Joel G. Breman, Martin S. Alilio, and Anne Mills. Conquering the intolerable burden of malaria: what's new, what's needed: a summary . Am. J. Trop. Med. Hyg., 71(2 suppl), 2004, pp. 1-15
r-1. World Health Organization, 2002. World Health Report. Reducing Risks, Promoting Healthy Life. Geneva: World Health Organization.
r-2. S. I. Hay, D. J. Rogers, J. F. Toomer and R. W. Snow. 2004. Annual P. falciparum Entomological Inoculation Rates (EIR) across Africa: Literature Survey, Internet Access, and Review Transactions of the Royal Society of Tropical Medicine and Hygiene 94: 113 - 27
r-3. The World Malaria Report 2005: Roll Back Malaria. Geneva: WHO.
r-4. Roberts, F., Roberts, C.W., Johnson, J.J., Kyle, D.E., Krell, T., Coggins, J.R., et al., (1998) Evidence for the shikimate pathway in apicomplexan parasites (published erratum appears in Nature 1998 Sep 17; 395(6699): 801; see comments). Nature 393: 801±805.
r-5. Craig W. Roberts, Fiona Roberts, Russell E. Lyons et al., The Shikimate Pathway and Its Branches in Apicomplexan Parasites. The Journal of Infectious Diseases. 2002;185 (Suppl 1):S25–36
r-6. Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402
r-7. Higgins D., Thompson J., Gibson T. Thompson J. D., Higgins D. G., Gibson T. J.(1994).
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice.
Nucleic Acids Res. 22:4673-4680
r-8. Galtier, N., Gouy, M. and Gautier, C. (1996) SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny. Comput. Applic. Biosci., 12, 543-548.
r-9. Puntervoll, P., Linding, R., Gemünd, C., Chabanis-Davidson, S., Mattingsdal, M., Cameron, S., Martin, D. M. A., Ausiello, G., Brannetti, B., Costantini, A., Ferrè, F., Maselli, V., Via, A., Cesareni, G., Diella, F., Superti-Furga, G., Wyrwicz, L., Ramu, C., McGuigan, C., Gudavalli, R., Letunic, I., Bork, P., Rychlewski, L., Küster, B., Helmer-Citterich, M., Hunter, W. N., Aasland, R., & Gibson, T. J. ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins.. Nucleic Acids Res. (2003), 31: 3625-3630.
r-10. Geourjon C, Deleage G . SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 1995 Dec;11(6):681-684
r-11. Silvio C.E. Tosatto. The Victor/FRST Function for Model Quality Estimation.
Journal of Computational Biology 2005; 12(10):1316-1327
r-12. Combet C, Jambon M, Deléage G & Geourjon C. Geno3D: Automatic comparative molecular modelling of protein. Bioinformatics, 2002, 18, 213-214
r-13. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 11, 2411-2423. [PubMed: 8563639]
r-14. Edelhoch, H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948-1954. [PubMed: 6049437]
r-15. Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326(1989). [PubMed: 2610349]
r-16. Bachmair, A., Finley, D. and Varshavsky, A. (1986) In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179-186. [PubMed: 3018930]
r-17. Gonda, D.K., Bachmair, A., Wunning, I., Tobias, J.W., Lane, W.S. and Varshavsky, A. J. (1989) Universality and structure of the N-end rule. J. Biol. Chem. 264, 16700-16712. [PubMed: 2506181]
r-18. Tobias, J.W., Shrader, T.E., Rocap, G. and Varshavsky, A. (1991) The N-end rule in bacteria. Science 254, 1374-1377. [PubMed: 1962196]
r-19. Ciechanover, A. and Schwartz, A.L. (1989) How are substrates recognized by the ubiquitin-mediated proteolytic system? Trends Biochem. Sci. 14, 483-488. [PubMed: 2696178]
r-20. Varshavsky, A. (1997) The N-end rule pathway of protein degradation. Genes Cells 2, 13-28. [PubMed: 9112437]
r-21. Guruprasad, K., Reddy, B.V.B. and Pandit, M.W. (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng. 4,155-161. [PubMed: 2075190]
r-22. Ikai, A.J. (1980) Thermostability and aliphatic index of globular proteins. J. Biochem. 88, 1895-1898. [PubMed: 7462208]
r-23. Kyte, J. and Doolittle, R.F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. [PubMed: 7108955]
r-24. R.G. Ridley, Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 415 (2002), pp. 686–693.
r-25. Glenn A. McConkey, John W. Pinney, David R. Westhead, Kirsten Plueckhahn, Teresa B. Fitzpatrick, Peter Macheroux and Barbara Kappes. Annotating the Plasmodium genome and the enigma of the shikimate pathway. Trends in Parasitology, Volume 20, Issue 2, February 2004, Pages 60-65

Author Information

Neelima Arora
Bioinformatics Group, Biology Division, Indian Institute of Chemical Technology

U.V.N. Mallikarjuna Chari
Bioinformatics Group, Biology Division, Indian Institute of Chemical Technology

Amit Kumar Banerjee
Bioinformatics Group, Biology Division, Indian Institute of Chemical Technology

U.S.N. Murty
Deputy Director/ Scientist “F”, Head, Biology Division, Indian Institute of Chemical Technology

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