Structure-function Relationship Of Weot-cleavage Product Hydrolases Based On Molecular Simulation Methods

Aromatics compounds have been widely used in various industries. Once they entry into the environment, it would cause the contamination of water, soil and air. Up to now, bioremediation using microbes appears to be the only viable option for large-scale removal of these contaminants from the environment. Meta-cleavage pathway, which exists broadly in the microbial catabolism of aromatic compounds, has a key hydrolytic step of upstream metabolism products. Meta-cleavage product (MCP) hydrolases which responsible for this step are often have strict substrate specificities, thus making this step become a critical bottleneck of the whole pathway. In this dissertation, the role of domain and non-active site residue were explored using MCP hydrolases from biphenyl biodegradation pathway, and the "structure blocks" for the function were clarified. Then, the catalytic promiscuity of MCP hydrolases were found, thus broadening the application range of these enzymes. Finally, two types of nanomaterial were employed to immobilize MCP hydrolases, in order to enhance the activity and stability in practical conditions.A MCP hydrolase encoding gene bphD was cloned from strain Dyella ginsengisoli LA-4, and the coding products were expressed in recombinant E. coli. BphD preferred to utilize HOPDA which possess a bulky phenyl substituent at C6atom, thus belonged to Group Ⅰ MCP hydrolase. The optimal temperature and pH were55℃and6.5, respectively. Three-dimensional structure obtained through homology modeling showed that BphD has the typical lid and core domain as the other α/β-fold hydrolases. The catalytic triad was Ser112-Asp237-His265. Molecule docking indicated that the steric hindrance and hydrogen bond interactions of the non-conserved residues in active pocket are important in determining the substrate specificity.The effects of different domains on substrate specificity were investigated using homologous recombination. The suitable crossover sites of BphD (Gly136and Ala211) and MfphA (Gly130and Alal96) were chosen through sequence energy analysis and molecular dynamic simulations. The substrate specificity of hybrid enzyme was similar to the parent enzyme which provided the lid domain, indicating the important role of lid domain. Further docking studies supposed an absence loop in Group II MCP hydrolases (corresponding to Ala208to Ser214in BphD) was the key structure element which affects the binding of HOPDA.The effect of non-active site residue in catalytic turnover was further explored. Metl48, which located at the entrance of tunnel, was chosen to mutagenesis according to the residue conserve analysis and tunnel identification.19mutants were then obtained through saturation mutagenesis. The catalytic efficiency (kcat/Km) of M148P decreased for74-fold, while this value for M148W which owing the most bulky side chain was almost the same with the wild-type. Molecular dynamic simulations shown that the corresponding tunnel in M148W had been blocked, thus this runnel was not the main tunnel for HOPDA transport. Meanwhile, the absence of two key hydrogen-bond interactions in M148P-HOPDA seemed responsible for the dramatic decrease of catalytic efficiency.Furthermore, catalytic promiscuity of MCP hydrolases was proven. BphD and MfphA could catalyze the hydrolytic reaction of p-nitrophenyl esters, and the catalytic efficiencies were in the same order of magnitudes with the natural C-C bond cleavage reaction. Large steric hindrance of phenyl moiety and improper hydrogen bond interaction made the poor reaction capacity of PNPBen with MfphA. On the other hand, BphD could also catalyze the hydrolytic reaction of TEOS, but the reaction did not depended on the catalytic triad, which was different with the hydrolytic mechanism of silicatein. Unexpectly, the synthesis capacity of NASICON type compound NaTi2(PO4)3was found in PBS buffer using BphD as template and catalyst. This reaction was also catalytic triad independent, but achieved through the interactions among positive surface residues and H2PO4-.Using protein titration curve and surface electrostatic distribution analysis, the optimal immobilization pH for MfphA and BphD were predicted, and then proved through adsorption kinetics. At the experimental conditions, the loading amount of MfphA on SBA-15was34mg/g, and the highest residual relative activity was25%when using different substrates. The loading amount of BphD was27mg/g, and no residual activity was detected. On the other hand, when pristine and carboxyl-modified CNTs were utilized as the immobilization matrix, the loading amounts promoted to276to508mg/g, which were more than10-fold of SBA-15. Meanwhile, BphD retained93.2%of initial activity when immobilized with carboxyl-modified SWCNT. However, SWNT could inhibit the activity of MfphA completely. The molecular dynamics simulation result of SWCNT-MfphA suggested a novel tunnel-blocking inhibition mechanism.