Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/14942
Authors: Singh, Neha
Keywords: Xenobiotic Compounds;Phthalic Acid Esters;Dimethyl Phthalate;Diethyl Phthalate;Polychlorinated Biphenyls
Issue Date: Apr-2018
Publisher: IIT Roorkee
Abstract: Xenobiotic compounds are man-made chemicals that are present in the environment at unnaturally very high concentrations. One such xenobiotic compound phthalates or phthalic acid esters (PAEs), are the dialkyl or alkyl aryl esters of o-phthalic acid, which are ubiquitous environmental pollutants. They are frequently used as plasticizers to provide stability to the plastic products. They have different properties which certainly depend on the composition and the type of alcohol that usually makes up the alkyl chain of phthalates. Several experimental and in silico molecular docking studies have highlighted the toxic effects related to phthalate exposure. This may eventually lead to the inhibition of normal activities of various receptors such as peroxisome proliferator-activated receptors, glucocorticoid receptors, estrogen receptors and progesterone receptors and etc. Six PAEs namely Dimethyl phthalate (DMP), Diethyl phthalate (DEP), Di-n-butyl phthalate (DnBP), Butyl benzyl phthalate (BBzP), Bis-(2-ethylhexyl) phthalate (DEHP) and Di-n-octyl phthalate (DnOP) are in the priority pollutants list of United States Environmental Protection Agency (USEPA) and European Union (EU) due to their explored toxicological, teratogenic and mutagenic properties. Likewise, polychlorinated biphenyls (PCBs) are groups of man-made organic chemicals consisting of carbon, hydrogen and chlorine atoms. The number of chlorine atoms and their location in a PCB congener determines many of its physical and chemical properties. Due to their non-flammability, chemical stability, high boiling point and electrical insulating properties, PCBs were extensively used in hundreds of industrial and commercial applications. Polychlorinated biphenyls (PCBs) are among the most persistent chlorinated environmental pollutants despite long-term regulation of their manufacture and use. The discovery of many bacterial strains that are able to partially degrade PCBs has fueled research directed towards improving the bioremediation strategies for the clean-up of PCB contaminated site. Keeping in mind the toxic effects of such harmful xenobiotic compounds and understanding the emergence of underlying concern; related to their toxicological aspect, to combat them with newer the bioremediation strategies, integrated structure based approaches have been used in the thesis objectives. Chapter 1 gives the brief introduction about the xenobiotic compounds. It further illustrates the use of microbial activity based bioremediation strategy to combat the continuing problem of xenobiotic pollution. Chapter 2 deals with the overview of computational approaches used for assessing the binding of commonly used high molecular weight phthalates such as dicyclohexyl phthalate (DCHP) and its monophthalate metabolite monocyclohexyl phthalate (MCHP), to the important human glucocorticoid receptor (hGR). The rise in rates of metabolic disorder is ultimately related to increase in exposure, usage, and production of these xenobiotic compounds. A large variety of environmental endocrine disrupting substances influence adipogenesis and obesity. “Obesogens” are chemical agents that improperly regulate the genes involved in glucose metabolism and adipocyte differentiation and promote lipid accumulation and adipogenesis. The human glucocorticoid receptor (hGR) is a steroid hormone triggered transcriptional factor and regulates target genes important in basal glucose homeostasis. Molecular docking analysis was performed in order to assess the in-silico structure based toxic effects of high molecular weight phthalate dicyclohexyl phthalate (DCHP) and its monophthalate metabolite mono-cyclohexyl phthalate (MCHP). DCHP and MCHP were docked within the active site cavity of the human glucocorticoid receptor (hGR). Molecular docking results show that the binding affinities of DCHP and MCHP lie within the comparable range with Dexamethasone (DEX), a potent agonist for hGR. Molecular Docking and simulation results emphasize that DCHP and MCHP can efficiently bind to hGR, which further leads to glucocorticoid-mediated adipogenesis in a synergistic manner. Chapter 3 explains the binding of phthalates with human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (hACMSD), a zinc-containing amidohydrolase which is a vital enzyme of the kynurenine pathway of tryptophan metabolism. It prevents the accumulation of quinolinic acid (QA) and helps in the maintenance of basal Trp-niacin ratio. To assess the structure based inhibitory action of PAEs such as DMP, DEP, DBP, DIBP, DEHP and their metabolites, these were docked into the active site cavity of hACMSD. Docking results show that the binding affinities of PAEs lie in the comparable range with Dipicolinic acid, a substrate analog of hACMSD. PAEs interact with the key residues such as Arg47 and Trp191 and lie within the 4 Å vicinity of zinc metal at the active site of hACMSD. Dynamics and stability of the PAEs-hACMSD complexes were determined by performing molecular dynamics simulations using GROMACS 5.14. Binding free energy calculations of the PAEs-hACMSD complexes were estimated by using MMPBSA method. The results emphasize that PAEs can structurally mimic the binding pattern of tryptophan metabolites to hACMSD, which further leads to inhibition of its activity and accumulation of quinolate in the kynurenine pathway of tryptophan metabolism. Chapter 4 describes the isolation and characterization of three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 for their degradative potential. These strains efficiently degraded 77.4% - 84.4% of DMP, 75.0% - 75.7% of DEP and 71.7% - 74.7% of DEHP, provided the initial amount of each phthalate is 500 mg L-1, after 44 hours of incubation. GC–MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA (phthalic acid). The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as mono-ethylhexyl phthalate (MEHP), mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show that the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3 Å, which is similar to the enzyme-substrate (ES) complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of phthalate monoester metabolites to phthalate. Chapter 5 describes the characterization of phthalate dioxygenase reductase (RePDR), an important enzyme of the PDO-PDR system from Ralstonia eutropha strain CH34, a gram-negative bacteria important from remediation viewpoint. PDR from Ralstonia eutropha strain CH34 was cloned, expressed and purified and characterized. The crystal structure of phthalate dioxygenase reductase is available from Burkholderia cepacia, a gram-negative bacterium (PDB ID: 2PIA). PDR has been reported to have three domains, the N-terminal FMN binding domain, central NAD (H) binding domains, and C- terminal [2Fe-2S] domain. The overall stability of the model of RcPDR was assessed by simulation studies and compared with 2PIA. In silico analysis of the complex formation of (phthalate dioxygenase), RePDO-RePDR is also studied and important interactions involved in complex formation were analyzed. Important interface residues from both the individual counterparts, i.e. RePDO and RePDR have been elucidated. The evolutionary relationship between important interface residues has also been established. Chapter 6 describes the screening of polychlorinated biphenyl (PCB) library with native Biphenyl dioxygenase (BPDO) structure from Pandoraea pnomenusa B-356, in order to evaluate the influence of the pattern and degree of chlorination on the catalytic activity of BPDO. Therefore, for that purpose, PCB congener library of 209 congeners was made and screened to elucidate the enhanced degradation abilities of BPDO. The important features related to congener specificity were highlighted. Through docking studies the binding ability of BPDO from Pandoraea pnomenusa B-356 w.r.t. PCB congeners was measured and important active site residues involved in the interaction were acknowledged. Moreover, the geometrical properties of the congeners such as aromatic benzene dimer ring offset, and the angle of rotation in between the two aromatic biphenyls influencing the rate of catalysis is also highlighted. The results related to the geometry and electrostatic properties of chlorinated biphenyls can be useful to rationalize their selective toxicities. Furthermore, structure determination of biphenyl dioxygenase from Pandoraea pnomenusa B-356 in complex with 2,3’,5’trichlorobiphenyl was also done.
URI: http://localhost:8081/xmlui/handle/123456789/14942
Research Supervisor/ Guide: Kumar, Pravindra
metadata.dc.type: Thesis
Appears in Collections:DOCTORAL THESES (Bio.)

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