Theoretical Studies On Structure Prediction, Docking And Catalytic Mechanism Study For Several Proteins

The 3D structure of protein and its ligand is important for clarifying its function and reaction mechanisms and it is useful for drug design.To date,X-ray diffraction or NMR techniques have been the most valuable tools for determining the 3D structures of biological molecules . Although the application of these experimental methods is continually growing,they remain time-consuming and limited applicability due to difficulties of accurate experimental data. With the rapid development of computer technology, computer aided molecular modeling has become a very important and active research field in chemistry, biochemistry and molecular biology. In this thesis, molecular dynamics simulations, quantum chemical calculation methods are used to build three dimensional models for seven proteins and study their structure properties in detail. Some creative results were obtained from the methods mentioned above. The main results are outlined as follows: 1. Homology modeling and molecular dynamics studies on the tomato methyl jasmonate esterase.Jasmonates are important in intracellular regulators mediating diverse developmental processes, such as seed germination, flower and fruit development, leaf abscission, and senescence. Jasmonic acid (JA) and methyl jasmonate esterase (MJE) are plant volatiles that induce plant defense responses against a group of pathogens and mechanical or herbivorous insect-driven wounding.The three-dimensional (3D) model of methyl jasmonate esterase (MJE), which is only responsible for methyl jasmonate (MeJA)-cleaving activity, is constructed based on the crystal structure of salicylic acid-binding protein 2 (SABP2, PDB code 1XKL) by using InsightII/Homology module, and further refined using unrestrained dynamics simulations. With the aid of the molecular interaction analysis between the natural substrate (MeJA), a 3D model of the complex MeJA-MJE is developed by molecular docking program, and the result may be helpful for further experimental investigations and the new mutant designs as well. In addition, the key binding-site residues are Thr107 and Leu214 which play an important role in the catalysis of MJE. Our results show that the identification of the theortical key binding site residues (Ser83 ), which plays an important role in the catalysis of MJE, is in consistent with experimental observation. The inhibitor phenylmethanesulfonyl fluoride is docked to MJE. Our results also show that His240 and His82 are important in inhibition and it may be helpful for the future inhibitor study. 2. Toward a blueprint forβ-Primeverosidase from tea leaves structure/function properties: Homology modeling studyFloral tea aroma is one of the most important factors to determine the character and quality of tea, especially oolong tea and black tea. Fresh tea leaves are virtually odorless or slightly smell of green note, and most of floral aroma compounds are produced by endogenous enzymes during tea manufacturing process of withering, rolling, and fermentation. Aβ-primeverosidase (EC 3.2.1.149) which is capable of hydrolyzingβ-primeveroside into a primeverose unit was reported.By means of the Homology modeling and the known structure of cyannogenicβ-glycosidase from white clover (1CBG, EC 3.2.1.21), we construct a 3D model of theβ–primeverosidase (EC 3.2.1.149) and further refined using molecular dynamics simulations. Furthermore, the docking of the substrates into the active site of the protein indicates thatβ-primeverosidase is able to hydrolyzeβ-primeverosides, but not acts on 2-phenylethylβ-D-glucopyranoside. These results suggest thatβ–primeverosidase shows broad substrate specificity with respect to the disaccharide glycon moiety (subsite -2). This is consistent with experimental observation. Thr271 and Thr415 play an important role in subsite -2 ofβ–primeverosidase. Our results may be helpful for further experimental investigations.3. Computational studies on bergaptol O-methyltransferase from Ammi majus L.: The substrate specificityFuranocoumarins compounds play an important role in plant growth and development as well as the interactions of plants with their environment. They come in a variety of flavors and have adverse affects on wide variety of organisms, ranging from bacteria to mammals. Some of the furanocoumarins are photoactive and their toxicity are enhanced in the presence of ultraviolet radiation and thence some of them are used to cure skin diseases.The primary sequence of bergaptol O-methyltranferase from Ammi majus L. (BMT) shows 65% homology with COMT. In order to understand the mechanisms of substrate specificity and the interaction between bergaptol and bergaptol O-methyltransferase (BMT), a 3D model of BMT is generated based on the crystal structure of caffeic acid 3-O-methyltransferase (COMT EC 2. 1. 1. 68 PDB code 1KYZ) by using the InsightII/Homology module. With this model, a flexible docking study is performed and the results indicate that BMT has narrow substrate specificity. Although the homology between both proteins is higher than 65% and all amino acids surrounding the binging site, except four residues, are similar in their sequences, the two proteins exhibit different substrate preferences. The differences in substrate specificity can be explained on the basis of the structures of the protein and the substrate. Our results indicate that His259 may be the catalytic base for the reaction, and Glu320, Glu287 bracket the catalytic His259. Especially, Glu320 forms a weak hydrogen bond with His259 and promotes the transfer of an H ion.4. The Three-dimensional structure of Human Aurora-C kinase predicted by homology modelingAurora-C is a key member of a closely related subgroup of serine/threonine kinase that plays an important role in the completion of essential mitotic events. By means of the homology modeling and the known structure of aurora-B, the 3D structure of aurora-C sourced human sapiens is constructed and then refined by using molecular mechanics (MM) optimization and molecular dynamics (MD) simulation. And then, the inhibitors H-89 and H-8 are docked to aurora-C. The docking study shows that Ala149 and Lys134 are important in inhibition as they form hydrogen bonds and have strong nonbonding interaction with H-89. We also suggest that Ile133, His130, and Ile148 are three important residues in binding as they have strong nonbonding interaction with H-89. The high affinity of H-89 compared with H-8 is explained by the much larger value of van der Waals energy with the enzyme.5. Homology modeling and docking studies of furcatin hydrolaseIt is known that many plant -glucosidases play a role in defending against pathogen attacks or injury by producing toxic aglycones Furcatin hydrolase (FH). FH is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O- -D-apiofuranosyl--D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose.The three dimension structure of furcating hydrolase (FH) was constructed by using homology modeling and mlecular dynamics methods. On the basis of the modeling, the components and structure of active site in FH were dentified. The docking of furcatin with FH has been performed, and the results show that the residues Ser84,Arg146,Thr189,Thr234,and Gly372 are important in binding of the complex. Ser84,Arg146,and Thr189 are important residues for subsite -1 for the disaccharide binding pocket in the active site of FH. Thr234 and Gly372 are important residues for subsite -2.6. On the 3D structure and catalytic mechanism study of amidase from Rhodococcus.erythropolisSome bacteria can use short-chain aliphatic amides as sources of nitrogen for growth by virtue of their ability to hydrolyze these amides to ammonia and the corresponding organic acid, using an amidase (EC 3.5.1.4). These enzymes are initially described in bacteria of environmental origin.The 3D structure of the amidase from Rhodococcus erythropolis (EC 3.5.1.4) is built by homology modeling. The docking studies show that Cys144 and Glu374 play an important role in substrates binding, and propionamide has stronger binding than that of acetamide. The computation models are set up to characterize explicit enzymatic reaction. The calculated free energy barrier at B3LYP/6-31G* level of model A (Ser194+propionamide) is 19.72 kcal?mol-1 in gas (6.47 kcal?mol-1 in solution), and model B (Ser194+Gly193+propionamide) is 18.71 kcal?mol-1 in gas (4.57 kcal?mol-1 in solution). Hydrogen-bonding interactions decrease the free energy barrier and control the orientation of the next reaction. Our results demonstrate that Ser194 is essential for acyl-intermediate, and Gly193 plays a secondary role in the stabilizing acyl-intermediate formation as the NH groups of Ser194 and Gly193 form hydrogen bonds with the carbonyl oxygen of propionamide. This is in good agreement with those derived from site-directed mutagenesis. 7. On the 3D structure and catalytic mechanism study of AmiF formamidase of Helicobacter pylori The human pathogen Helicobacter pylorus is a microaerophilic, spiral-shaped, Gram-negative bacterium that colonizes the gastric mucosa. It is a risk factor for the development of gastric cancer. This pathogen produces several virulence factors. One of the major factor contributing to acid resistance of H. pylori is the production of ammonia by its urease enzyme, which is essential for gastric colonization in different animal models. Urea is thought to be the main source of ammonia in the gastric environment, but H. pylori does have alternative pathways for the production of ammonia via amino acid catabolism and for the activity of its two paralogous amidases, aliphatic amidase (AmiE, EC 3.5.1.49) and formamidase (AmiF(fhp) EC 3.5.1.49).The 3D structure of the AmiF formamidase of Helicobacter pylori (denoted as AmiF(fhp)) is built by homology modeling. The docking studies show that AmiF(fhp) has restricted substrate specificity, as it only hydrolyzes formamide. In order to reveal the reaction mechanism and the catalytic role for Cys166, Lys133 and Asp168 in AmiF(fhp), three quantum chemical calculation models are constructed based on the 3D structure of AmiF(fhp) and the reaction paths are obtained at B3LYP 6-31+G* level. The calculated results show that (1) the reaction of Cys166-formamide anion in the enzyme active proceeds via a transition state without the intervention of tetrahedral intermediate. (2) the positive charge on Lys133 polarizes the formamide in the transition state region to redistribute the electron and thence decrease the free energy barrier. (3) the active site residue of Asp168 increases the free energy barrier as the negative charge will affect the electron distribution.