N Arora, U Chari, A Banerjee, U Murty
chorismate synthase, malaria, shikimate pathway
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.
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
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
Sequence analysis of Chorismate Synthase (CS) of
A phylogenetic tree was constructed with the help of PHYLO_WIN tool (Galtier
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
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
Functional Characterization of CS: Functional characterization of CS protein sequence was done by finding motif using Eukaryotic Linear Motif (ELM tool (
Structure prediction of
Secondary structure prediction: SOPMA tool (Geourjon and Deleage, 1995)(
3D structure prediction: A comparative 3D structure analysis of
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.
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
Further, CS protein was subjected to CLUSTALW for multiple sequence alignment and phylogenetic analysis (Fig. 1). The result suggests that CS protein of
A phylogenetic tree was constructed using Neigbour Joining method (NJ) with100 bootstrap and 500 bootstraps replicate by using PHYLO_WIN. (Fig. 2).
# 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
Secondary structure prediction:
SOPMA program that was used to predict secondary structures in
Three dimensional structure of CS of
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.
Other web based servers were also employed to generate the 3 dimensional structure of
Based on these evaluations, we conclude that model obtained using HOMER is superior as compared to models generated using other web based servers.
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
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.
U.S.N Murty Deputy Director/ Scientist “F” Head, Biology Division, Indian Institute of Chemical Technology, Hyderabad-500007, India. Email: email@example.com Phone: +91 40 27193134; Fax: +91 40 27193227