The Preliminary Study On The Cold Adaptation Mechanism Of 2,4-Dichlorophenol Hydroxylase TfdB-JUL

There is a continuous increase in environmental pollution because of human activities,bioremediation has become the preferred method for environmental restoration due to its low cost,low energy consumption,and environmental friendliness.Most of the earth’s surface is in a low temperature environment,but bioremediation in low temperature environment generally has problems of low efficiency,long cycle,high energy consumption and high cost,which is a worldwide problem in the field of environmental protection.Cold-adapted enzymes are valuable tools for bioremediation in low temperature environments due to their inherent properties such as low catalytic optimum temperature,high catalytic efficiency,and thermal instability.Our group previously discovered and reported a new 2,4-dichlorophenol hydroxylase Tfd B-JLU with cold-adaptive activity,which is involved in the internationally widely used herbicide 2,4-dichlorophenoxyacetic acid(2,4-D)is a key enzyme in the degradation reaction,but the cold adaptation mechanism of this enzyme is still unclear.The purpose of this paper is to explore the structural characteristics of Tfd B-JLU through the analysis of its sequence and structure,molecular docking and molecular dynamics simulation,combined with rational design,and to explore its thermodynamic changes through the study of enzyme molecular thermodynamics and kinetics.The relationship between the law and cold adaptability,based on the above studies,the molecular mechanism of the cold adaptation of Tfd B-JLU enzyme was preliminarily analyzed.The main contents and findings of this study are as follows:(1)construction and evaluation of Tfd B-JLU structure;structure is the basis of function,determines the biological function of protein,and is also the premise to study the mechanism behind the function.Since the crystal structure of Tfd B-JLU has not been resolved,it has certain limitations to study its cold adaptation mechanism.In this study,Alpha Fold2 was used to construct the structure of Tfd B-JLU,and the constructed protein structure was evaluated by Ramachandran plot,Profiles-3D and Z-score values.The results showed that our constructed protein structure was highly credible(2)Structure-based site prediction and verification;by summarizing the structural features of several cold-adapted enzymes,it was found that the structural characteristics of cold-adapted enzymes related to cold-adaptation vary from enzyme to enzyme,and it is necessary to compare the sequence and structure with the homologues of thermophilic or thermophilic enzymes.In order to explore the structural features of Tfd B-JLU related to cold adaptation,Tfd B-JLU and two mesophilic Tfd B enzymes(respectively from Cupriavidus necator JMP134,which is the most studied and in-depth,and Cupriavidus oxalaticus,which is highly homologous to Tfd B-JLU)were compared for structural alignment and molecular dynamics simulation studies.It was found that Tfd B-JLU was more flexible than the two mesothermal homologous enzymes.It was found that the high flexibility of TFDB-JLU was closely related to the decrease of salt bridge,hydrogen bond,Arg,Asp,surface charge,Arg/Lys ratio and the proportion of charged and polar uncharged amino acids,and the higher proportion of small amino acids and neutral amino acids as well as the higher proportion of surface hydrophobic amino acids and more long loop regions.Based on the structural characteristics of TFDB-JLU flexible structure,the amino acids that may play a key role in TFDB-JLU cold adaptation were preliminarily predicted,the mutation library was constructed and the mutation strategy was formulated.The correlation between these sites and cold adaptation was explored through rational design verification.The results showed that the cold adaptation of L249 G and P316 G was further improved,and the cold adaptation of Q423 was decreased as expected.It was speculated that L249,P316 and Q423 might be related to the cold adaptation of TFDB-JLU.(3)Thermodynamics and cold-adaptive mechanism of Tfd B-JLU;the high catalytic efficiency of cold-adaptive enzymes at low temperature benefits from its unique cold adaptation mechanism.In order to explore the cold adaptation mechanism of Tfd B-JLU,we conducted thermodynamic studies on mutants with better cold adaptation by means of bioinformatics and data measurement.The mutants all showed a trend of decreasing ?H? and increasing ?S?,which is consistent with the change trend of basic thermodynamic characteristics of other cold-adapted enzymes.At the same time,the change of enthalpy and entropy increased the substrate confusions of L249 G,M251G and M251G/P316 Q mutants,which may be characterized by the coevolution of substrate non-specificity and cold adaptation.The kinetic parameters of the mutants M251 G and M251G/P316 Q were determined,and it was found that the affinity of the mutants M251 G and M251G/P316 Q to 2,4-DCP was significantly higher than that of the wild type.In conclusion,the cold-adaptive mechanism of Tfd B-JLU may be mainly related to the amino acids in the active center region.Combined with the analysis of thermodynamic characteristics and structural characteristics,the cold adaptation of TFDB-JLU may be mainly regulated by amino acids in the active center and active pocket region,and the distal region has little influence on the cold adaptation.The enzyme may have evolved its cold adaptation by increasing the local structural flexibility,reducing the energy consumption in the reaction process,and improving the low temperature catalytic efficiency by entropy enthalpy compensation.To sum up,this paper predicts and evaluates the structural features related to the cold adaptation of Tfd B-JLU,and preliminarily grasped some structural features related to its cold adaptation.Combined with the study of thermodynamic characteristics,a preliminary understanding of Tfd B-JLU’s cold adaptation mechanism was obtained,which laid an important foundation for the final analysis of its mechanism.This study is of great significance for the evolution of cold-adapted enzymes and the enrichment and development of bioreremediation of aromatic pollutants in cold regions.