Sujata Sharma

All India Institute of Medical Sciences, New Dilli, NCT, India

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Publications (101)355.75 Total impact

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    ABSTRACT: During the course of protein synthesis in the cell, the translation process often gets terminated due to various reasons. As a result of this, peptidyl-tRNA molecules are released which are toxic to the cell as well as it reduces the availability of free amino acid and tRNA molecules for the required protein synthesis in the cell. Such a situation is corrected by an enzyme, peptidyl-tRNA hydrolase (Pth) which catalyses the release of free tRNA and peptide moieties from peptidyl-tRNAs. This means that the active Pth is essential for the survival of bacteria. In order to design inhibitors of Pth from Pseudomonas aeruginosa (PaPth), we have determined the structures of PaPth in its native state and in the bound states with compounds, (i) amino acylate-tRNA analogue and (ii) 5-azacytidine. The structure determination of native protein revealed that the substrate binding site was partially occupied by Glu161 from the neighbouring molecule. Structure of PaPth indicated that the substrate binding site can be broadly divided into three distinct subsites. The structures of the two complexes showed that the compound (i) filled three subsites whereas compound (ii) filled two subsites. The common sugar and the base moieties of the two compounds occupied identical positions in the cleft. Using Surface Plasmon resonance, the dissociation constants for compounds (i) and (ii) were found to be 3.53×10-8 M and 5.82 ×10-8 M respectively.
    The Biochemical journal. 08/2014;
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    ABSTRACT: Bovine lactoferrin, a 76 kDa glycoprotein (Ala1 - Arg689) consists of two similar N- and C-terminal molecular halves with ability to bind two Fe3+ ions. The N-terminal half, designated as N-lobe (Ala1 - Arg341) and C-terminal half designated as C-lobe (Tyr342 - Arg689) have similar iron binding properties but the resistant C-lobe prolongs its physiological role in the digestive tract. Here we report the crystal structure of true C-lobe which was produced by limited proteolysis of bovine lactoferrin using trypsin. In the first step of proteolysis, two fragments of 21kDa (Glu86 - Lys282) and 45kDa (Ser283 - Arg689) were generated because two lysine residues, Lys85 and Lys282 in the structure of iron-saturated bovine lactoferrin were fully exposed. The 45kDa fragment got further digested at the newly exposed side chain of Arg341 generating a 38kDa perfect C-lobe (Tyr342 - Arg689). On the other hand, the apolactoferrin was cut by trypsin only at Arg341 which was exposed in the structure of apolactoferrin while the other two sites with Lys85 and Lys282 are inaccessible. The purified iron saturated C-lobe was crystallized at pH 4.0. The structure was determined with molecular replacement method using coordinates of the C-terminal half (Arg342 - Arg689) of the intact camel apolactoferrin. The structure determination revealed that the iron atom was absent and the iron-binding cleft was found in a wide open conformation whereas in the previously determined structure of iron saturated C-lobe of bovine lactoferrin, iron atom was present and the iron-binding site was in the closed confirmation.This article is protected by copyright. All rights reserved.
    FEBS Journal 05/2014; · 4.25 Impact Factor
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    ABSTRACT: Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.
    Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
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    ABSTRACT: Polo-like kinase (Plk)1 is a key regulator of the cell cycle during mitotic phase and is an attractive anti-mitotic drug target for cancer. Plk1 is a member of Ser/Thr kinase family which also includes Plk2-4 in human. Plk1 promotes the cell division whereas Plk2 and Plk3 are reported to act as tumour suppressors. The available inhibitors of Plk1 also suppress Plk2 and Plk3 activity significantly resulting in the cell death of normal cells in addition to the cancer cells. Hence, it is imperative to explore Plk1 specific inhibitors as anti-cancer drugs. In this work, a selective potential inhibitor of Plk1 has been identified by molecular docking based high throughput virtual screening. The identified compound exploits the subtle differences between the binding sites of Plk1 and other Ser/Thr kinases including Plk2-4. The predicted binding affinity of identified inhibitor is higher than available inhibitors with a 100-fold selectivity towards Plk1 over Plk2-4 and several cell cycle kinases. It also satisfies the Lipinski's criteria of drug-like molecules and passes the other ADMET filters. This triazole compound with aryl substituent belongs to a novel class of potential inhibitor for Plk1. The suggested potential lead molecule can thus be tested and developed further as a potent and selective anti-cancer drug.
    Journal of Molecular Graphics and Modelling. 01/2014;
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    ABSTRACT: Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of Peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014; · 3.73 Impact Factor
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    ABSTRACT: Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.
    PLoS ONE 01/2014; 9(3):e90011. · 3.73 Impact Factor
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    ABSTRACT: Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.
    The Scientific World Journal 01/2014; 2014:543195. · 1.73 Impact Factor
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    ABSTRACT: Trypanosoma brucei Pteridine reductase (TbPTR1) is of vital importance and is an established drug target for dreaded Human African trypanosomiasis (HAT). Pharmacophore perception strategy has been employed to identify key chemical features responsible for the biological activity for TbPTR1. The findings suggest that three different pharmacophore features can be associated with T. brucei anti‐PTR1 activity namely: H‐bond donors (D), Hydrophobic aromatic (H) and Ring aromatic (R). The resulting hypothesis is able to predict the activity of other existing TbPTR1 inhibitors with a correlation coefficient (r) of 0.89. An in silico database screening, based on the best hypothesis, has been used to identify some potential nanomolar range TbPTR1 inhibitors. These compounds were then checked by molecular docking and subjected to ADMET analysis. Further, a detailed comparison of the pharmacophore behavior and differential analysis of binding pockets of T. brucei and L. major was made which revealed subtle differences in terms of their shape and charge properties. This investigation can form the basis for tweaking the specificity of compounds for generating new improved species specific inhibitor molecules for Pteridine reductase in these different parasitic protozoans.
    Molecular Informatics 01/2014; 33(2). · 2.34 Impact Factor
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    ABSTRACT: Acinetobacter baumannii is a virulent pathogenic bacterium that is resistant to most currently available antibiotics. Therefore, the design of drugs for the treatment of infections caused by A. baumannii is urgently required. Dihydrodipicolinate reductase (DHDPR) is an important enzyme which is involved in the biosynthetic pathway that leads to the production of L-lysine in bacteria. In order to design potent inhibitors against this enzyme, its detailed three-dimensional structure is required. DHDPR from A. baumannii (AbDHDPR) has been cloned, expressed, purified and crystallized. Here, the preliminary X-ray crystallographic data of AbDHDPR are reported. The crystals were grown using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitating agent The crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 80.0, b = 100.8, c = 147.6 Å, and contained four molecules in the asymmetric unit. The complete structure determination of AbDHDPR is in progress.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2013; 69(Pt 6):653-6. · 0.55 Impact Factor
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    ABSTRACT: Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ -phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography.
    Enzyme research. 01/2013; 2013:597028.
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    ABSTRACT: Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.
    International journal of biochemistry and molecular biology. 01/2013; 4(2):95-101.
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    ABSTRACT: Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.
    International journal of biochemistry and molecular biology. 01/2013; 4(3):108-128.
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    ABSTRACT: Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A-B and C-D. The A-B and C-D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A-B and C-D contacts. Two ligand binding sites, one at C-D contact and another at A-B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both Gram-positive and Gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C-D contact (Site-1) while SA binds to it at the A-B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.
    PLoS ONE 01/2013; 8(1):e53756. · 3.73 Impact Factor
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    ABSTRACT: Lactoferrin is an iron-binding diferric glycoprotein present in most of the exocrine secretions. The major role of lactoferrin, which is found abundantly in colostrum, is antimicrobial action for the defense of mammary gland and the neonates. Lactoferrin consists of two equal halves, designated as N-lobe and C-lobe, each of which contains one iron-binding site. While the N-lobe of lactoferrin has been extensively studied and is known for its enhanced antimicrobial effect, the C-lobe of lactoferrin mediates various therapeutic functions which are still being discovered. The potential of the C-lobe in the treatment of gastropathy, diabetes, and corneal wounds and injuries has been indicated. This review provides the details of the proteolytic preparation of C-lobe, and interspecies comparisons of its sequence and structure, as well as the scope of its therapeutic applications.
    Biochemistry research international. 01/2013; 2013:271641.
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    ABSTRACT: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most highly prescribed drugs in the world. Their analgesic, anti-inflammatory, and antipyretic actions may be beneficial; however, they are associated with severe side effects including gastrointestinal injury and peptic ulceration. Though several approaches for limiting these side effects have been adopted, like the use of COX-2 specific drugs, comedication of acid suppressants like proton pump inhibitors and prostaglandin analogs, these alternatives have limitations in terms of efficacy and side effects. In this paper, the mechanism of action of NSAIDs and their critical gastrointestinal complications have been reviewed. This paper also provides the information on different preventive measures prescribed to minimize such adverse effects and analyses the new suggested strategies for development of novel drugs to maintain the anti-inflammatory functions of NSAIDs along with effective gastrointestinal protection.
    Mediators of Inflammation 01/2013; 2013:258209. · 3.88 Impact Factor
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    ABSTRACT: The incidences of infections caused by an aerobic Gram-negative bacterium, Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.
    PLoS ONE 01/2013; 8(7):e67547. · 3.73 Impact Factor
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    ABSTRACT: Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)8 triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities.
    International journal of biochemistry and molecular biology. 01/2013; 4(4):166-78.
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    ABSTRACT: Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin.
    International Journal of Peptides 01/2013; 2013:390230.

Publication Stats

599 Citations
355.75 Total Impact Points

Institutions

  • 2001–2014
    • All India Institute of Medical Sciences
      • Department of Biophysics
      New Dilli, NCT, India
  • 2011
    • Panjab University
      • Department of Biotechnology
      Chandīgarh, Union Territory of Chandigarh, India
  • 2010
    • Sardar Patel University
      Vallabh Vidhyanagar, Gujarāt, India
  • 2009
    • Central Drug Research Institute
      • Molecular and Structural Biology Division (CDRI)
      Lucknow, Uttar Pradesh, India