Using Combinatorial Mutation To Regulate The Promiscuity And Cold Adaptation Of 2,4-dichlorophenol Hydroxylase

2,4-Dichlorophenoxyacetic acid(2,4-D)and 2,4-Dichlorophenol(2,4-DCP)have a wide range of uses in industrial and agricultural production processes,such as herbicides and antiseptics agents,which are used in large quantities.It has received extensive attention in recent years because of its high pollution and refractory degradation.Physical and chemical methods of degrading these chemicals generally have problems such as low conversion efficiency,high cost,and large secondary pollution.Biological methods have become a research hotspot in recent years due to its high efficiency,low cost and environmental protection.2,4-Dichlorophenol hydroxylase(tfd B enzyme)is a key enzyme involved in the 2,4-D degradation reaction.It performs the key step of oxidizing 2,4-DCP,and it is a promising enzyme in biodegradation of 2,4-D.However,tfd B enzymes obtained through various methods currently have problems such as low catalytic efficiency and poor environmental adaptation.In the early stage of our research group,the Tfd B-JLU gene was obtained by metagenomic extraction from the soil contaminated by PCBs for a long time,and the plasmid was constructed into E coli for exogenous expression,and the Tfd B-JLU enzyme was obtained.The enzyme has high catalytic activity,substrate promiscuity and excellent properties such as cold adaptability.As the crystal structure of Tfd B-JLU enzyme(2,4-dichlorophenol-6-monooxygenase)has not yet been obtained,the template enzyme used in the preliminary study of homology modeling by this research group is 2-hydroxybiphenyl-3-monooxygenase.Its PDB number is 5brt,and the identity is 41.97%.Pie E with PDB number 6u0 p has 46.55% similar identity to Tfd B-JLU,and the enzyme is also 2,4-dichlorophenol-6-monooxygenase,and its properties are more similar to Tfd BJLU.Therefore,in this study,we used 6u0 p as a template to model the Tfd B-JLU structure homologously through SWISS-MODEL.In order to clarify the structure-activity relationships and mechanism,this study intends to combine computer-aided design to regulate the above-mentioned properties of Tfd B-JLU enzyme through CASTing/ISM,which is a combination of iterative saturation mutagenesis(ISM)and combined active site saturation test(CASTing),which are widely used in directed evolution research.The research content of this subject mainly includes three aspects: The first one is to search for template enzymes through sequence alignment,and to construct homology modeling of the Tfd B-JLU enzyme structure;to study the interaction between Tfd B-JLU and small molecule ligands,key binding regions and amino acid sites through molecular docking.The second is the combination design of key sites through CASTing/ISM and directed evolution to regulate the catalytic activity,promiscuity and cold adaptation of the Tfd B-JLU enzyme.The third is to study the structure-activity relationships and mechanism of the enzyme through molecular dynamics simulation.The research results of this subject are as follows: We used 6u0 p as a template to model the Tfd B-JLU structure homologously through SWISS-MODEL,and evaluated the resulting structure.Through the Ramachandran plot,verify-3D and Z-score under the Procheck program,scores show that our model is credible.The constructed structure was used for molecular docking through Autodock4.2 software.By analyzing the interaction between Tfd B-JLU and small molecule ligands,the original key sites were confirmed,and we proposed new key amino acid sites that may have an effect on the properties of the enzyme.It is confirmed that M251 is the key amino acid position at the entrance of the active pocket.Then,we combined the mutation sites through CASTing/ISM and selected the three mutation sites H47 R,M251G,and P316 Q,which worked well in the previous experiments.We designed double-point mutations and three-point mutations H47R/M251 G,M251G/P316 Q,H47R/M251G/P316 Q,these three mutants.We successfully expressed and purified the target proteins.In terms of enzyme catalytic activity and promiscuity,we found that the two-point M251G/P316 Q mutant has a 2-fold increase in the specific activity of 2,4-DCP,and the specific activity of 3,5-DCP and 2,4,6-TCP has increased by49 and 41 times.The property of promiscuity has changed significantly.Through molecular docking,we found that the free energy of binding between mutant M251G/P316 Q and 3,5-DCP,2,4,6-TCP decreased.The enhancement of the binding ability may be the reason for the enhancement of its catalytic activity.In terms of the evolution of cold adaptation,by continuing to study the successfully constructed mutants at 4℃,25℃,and 37℃,we successfully found the specific activity of H47R/M251G/P316 Q increased by 11 times compared to the wild type at 4℃.Through molecular dynamics simulation methods,the reasons for the enzyme’s cold adaptation were analyzed.By simulating the structure of the enzyme at 4℃,25℃,and 37℃,it is found that at 4℃,there are two more alpha helices in the 71-74 and 178-181 amino acid residues,and the protein structure becomes more loose,and the reason why the enzyme is coldadaptable is further analyzed and explored.To sum up,this thesis improves the catalytic activity of Tfd B-JLU enzyme through CASTing/ISM combined mutation,which changes its promiscuity and cold adaptation,and uses computer simulation methods to further study its structure-activity relationships and mechanism,which provides an important research basis for the directed evolution of such enzymes.