Study On The Function Of Key Amino Acids In Substrate Binding Loop Of Thermus Thermophiles SG0.5JP17-16 Laccase

Laccase?EC1.10.3.2?is a polyphenol oxidase and the electron of substrate was abstracted at the T1 Cu site,then transferred through the electron transfer chain inside the laccase,finally arrived at the TNC to reduce O₂ to water.Laccase has the characteristics of low substrate specificity,which makes it useful in many fields,such as food industry,environmental protection and pulp and paper.Laccase is a green catalyst with excellent characteristics.The laccase lac TT produced by Thermus thermophiles SG0.5JP17-16 is a bacterial laccase with good resistance to p H and high temperature.Based on sequence comparison and structural analysis,five sites on the substrate binding loop that may have an effect on catalysis were predicted:E199,D289,R290,D394 and D396,then these five sites were mutated to Met,respectively.D394 and D396 residues were selected according to the changes in catalytic efficiency of mutants,and the role of the two sites in the catalytic process was studied.?1?Study on the function of D394 site.Studies on enzymatic properties showed that the optimal p H of the mutants decreased slightly,and the thermal stability decreased significantly.The kinetic data revealed that the catalytic efficiency of the D394E,D394M and D394R mutants reduced as compared to that of the wild type,while the catalytic efficiency of the D394N mutant enzyme was similar to that of the wild type enzyme.UV-Vis absorption spectrum data indicated that the absorption intensity of the D394E,D394M and D394R mutant enzymes was lower than that of the wild type at 608 nm,suggesting that the geometry structure of the T1 Cu site might be disturbed,which might further affect the mutants enzyme catalytic efficiency.Previous studies had revealed that T1 Cu ligand,H397,plays an important role in the electron transport process.Our structural analysis revealed that the hydrogen bonding network in wild-type and D394N mutant enzymes contained T1 Cu ligand residue H397,while for D394E,D394M,D394R mutant enzymes,the H397 residue was not in the hydrogen bonding network.Changes in the hydrogen bonding network might result in the reduced catalytic efficiency of D394E,D394M,and D394R mutant enzymes.CD spectra of the wild-type and D394R mutant enzyme showed that the secondary structure of the D394mutant changed.Combined with the structural data,the mutation from D394 to E394,M394,and R394 might change the configuration of the loop where the D394 residue is located.The redox potential results showed that the redox potential of the D394E,D394M,and D394R mutant enzymes were lower than that of the wild type,and the reduction of the potential might also lead to a decrease in the catalytic efficiency of mutants.In summary,the replacement of the D394 residue by E394,M394,and R394 residues might affect the configuration of the loop in which they are located,and change the hydrogen bonding network,the geometry structure and electric potential of the T1 Cu site,then further affect the catalytic efficiency of laccase.The decolorization experiment of D394R mutant and wild type enzymes showed that the decolorization rate of D394R mutant was similar to that of the wild type enzyme for azo dyes RBB,RBWNN and CR,and the decolorization rate of anthraquinone dye RBBR decreased significantly.?2?Functional study of D396 site.Enzymatic properties studies showed that the optimal p H of all mutants was all shifted to 5.0,and the optimal temperature and thermal stability were similar to those of the wild type enzyme.The catalytic efficiency of the D396A,D396E and D396M mutants increased as compared with that of the wild type enzyme,while the catalytic efficiency of the D396N mutant was similar to that of the wild type.The UV-visible spectrum data showed that the absorption peak intensity of D396A,D396E and D396M mutants changed at 608 nm,indicating that the geometry structure of the T1 Cu site might be disturbed.CD spectra of wild-type and D396M mutant enzymes showed that the secondary structure changed.Structural analysis showed that the wild-type and D396N mutant enzymes contained D396-Y288 and N396-Y288 hydrogen bonds at the substrate entrance,while in D396A,D396E,and D396M mutants,D396-Y288 and N396-Y288 hydrogen bonds disappeared,L427-H397 hydrogen bonds was formed.These changes might lead to the increased catalytic efficiency of D396A,D396E and D396M mutants.In addition,the reduced steric hindrance of the A396 residue was beneficial to substrate binding.In summary,the mutations from D396 to A396,E396 and M396 changed the hydrogen bonding network,steric hindrance and the geometry structure of the T1 Cu site,which in turn affected the catalysis of laccase.The decolorization experiment showed that the decolorization rate of the D396M mutant for RBB,RBWNN,CR was similar to that of the wild type enzyme,and the decolorization rate of RBBR by the D396M mutant was 7%higher than that of the wild type enzyme.