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Classification Of Mode Of Action Of Phenol Derivatives And The Effect Of Dioxygen Pathway On Their Biodegradation

Posted on:2010-05-09Degree:DoctorType:Dissertation
Country:ChinaCandidate:L XuFull Text:PDF
GTID:1101360302460655Subject:Pharmaceutical Engineering
Abstract/Summary:PDF Full Text Request
Phenols and their derivatives are ubiquitous environmental contaminants and toxic to many organisms by interfering energy transduction in cells. With different substitutions, phenols may exert various biological activities. Besides, one kind of toxicity may be caused by the superposition of effects from different mechanisms. Both classification (qualitative) and QSAR (quantitative) approaches have currently been applied to predict the toxicity of chemicals. One of fundamental issues is how to select and interpret the molecular descriptors used in both methods. Extensive applications of support vector machines (SVM) in classification and QSAR analysis have made it one of popular machine-learning methods in recent years. However, the number of phenols with known mode of action is small, whether SVM is applicable to such problem is worth investigating, as well as how to validate and interpret the classification results.The biodegradation of phenols and their derivatives is one of the important ways to reduce their contamination and toxicity. Dioxygenases play an important role in the biodegradation of catechol and its derivatives by catalyzing the cleavage of aromatic rings. Generally, the intradiol dioxygenases require Fe3+ to cleave C-C bond between the phenolic hydroxy groups to produce cis, cis-muconic acid, while the extradiol dioxygenases use Fe2+ (or Mn2+) as a cofactor to cleave the C-C bond adjacent to the phenolic hydroxy groups to yield 2-hydroxymuconaldehyde. Both experimental and theoretical investigations have been focused on the detection of intermediates in the reaction cycle in order to develop a general chemical mechanism of O2 activation and insertion. However, little is known about the mechanism of how O2 reaches the reaction sites of the related enzymes, which raises the question whether the rate of catalysis is limited by O2 access to the active site. The precise, atomic-resolution pathways for O2 migration in the protein, along with predicted relative significant parts of the pathways, should help to rationalize the selectivity of specific intermediate at the active site of each subunit and greatly facilitate the selection of specific site mutations for such studies.In this paper, the combination of SVM and molecular dynamics simulations enables us to investigate the classification of phenols and their derivatives from different scales. Two approximation methods, locally enhanced sampling and implicit ligand sampling, were applied to find the O2 pathways in the extrodioxygenase of 2,3-HPCD. These works are outlined as follows. 1. In this work, we first employed SVM on a dataset containing 155 polar narcotics and 19 uncouplers to filter the predictive hydrophobic and hydrogen bonding descriptors. The overall classifaction rate was above 99%. Molecular dynamics (MD) simulations were then conducted to investigate the behavior of salicylate (SAL) and pentachlorophenol (PCP) molecules in the context of a palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer. The results demonstrated that their equilibrium properties in the lipid bilayer were closely associated with hydrophobic and hydrogen bonding descriptors. The preferred position of SAL in the POPC bilayer lies in the lipid headgroup, while PCP resides in the region between carbonyl groups and water phase (lipid-water interface). SAL could form stable hydrogen bonds with carbonyl oxygen atom of oleoyl chain in POPC, as well as intermolecular hydrogen bonds. PCP acts as hydrogen acceptor and establishes hydrogen bonds mainly with water. The observations from molecular dynamics simulations facilitated to elucidate the mechanism of polar narcotics and uncouplers.2. The experimental determination of the structure of Fe2+-containing homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) with X-ray crystallography showed that three different intermediates reside in different subunits of a single homotetrameric enzyme molecule. In this paper, two locally enhanced sampling molecular dynamics simulations were performed to determine the potential O2 pathways inside a recently solved X-ray structure of homoprotocatechuate 2,3-dioxygenase. It is found that nominally identical subunits of the single homotetrameric structure contain distinct O2 affinity diffusion pathways, which partly correlates with the observation of the simultaneous presence of three different reaction intermediates in four independent active sites. Residues that are critical for O2 diffusion are also examined and discussed. In particular, we find that the breathing motion of internal cavity defined by these residues results in O2 migration process.3. Based on the trajectory of a 10-ns molecular dynamics simulation, implicit ligand sampling was applied to calculate the 3D free energy map for O2 inside the protein. The energetically optimal routes for O2 diffusing were identified for each subunit of the homotetrameric protein structure. The O2 tunnels formed due to thermal fluctuations were also characterized by connecting elongated cavities inside the protein. Superimposing the favorable O2 tunnels onto the free energy map, both energetically and geometrically preferred O2 pathways were determined, as well as the amino acids that may be critical for O2 passage along these paths. Our results demonstrate that identical subunits possess quite distinct O2 tunnels. The order of O2 affinity of these tunnels is generally consistent with the order of catalytic rate of each subunit. As a consequence, the subunit containing the highest O2 affinity pathway has the highest probability for finding product of reaction. Compared with results of LES, we find that it is the frequency of the breath motion of the cavity that limits O2 acess to the actie site of each monomer. In contrast to randomized behavior, O2 diffusion in the four subunits is clearly limited to specific regions located within the conserved active site domain.In summary, the combination of SVM and MD simulations provides a new strategy for elucidating the mechanism of toxic action underlying the corresponding molecular descriptors. The indentifation of O2 pathways in 2,3-HPCD will be valuable to engineer non-heme iron dioxygenases in order to find intermediates by charactering O2 diffusion routes.
Keywords/Search Tags:Classification, Molecular Dynamics, Dioxygen pathway, Locally enhanced sampling, Impicit ligand sampling
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