The Regulation Mechanism Of Key Loops Toward The Stability And Activity Of ?-glucuronidase

?-Glucuronidase(GUS)has been widely used in modifying natural glucuronides compounds to improve their activity and bioavailability.The previous work in our lab have identified a GH2?-glucuronidase which was derived from Aspergillus terreus Li-3(PGUS),and PGUS can hydrolyze natural substrate GL(glycyrrhizin)into more valuable GA(glycyrrhetinic acid).However,the poor stability,low activity and the unclear catalytic mechanism of PGUS in hydrolyzing GL severely limited the engineering modification and industrial application.In this research,several key loops influencing the stability and activity of PGUS were found through the combination of structure analysis and computer simulation.We rationally designed and deleted the C-terminal loop of PGUS to improve its catalytic ability,meanwhile,we also explored the molecular mechanism of PGUS in hydrolyzing GL with focus on two key loops:Floop and loop470.The main results are as follows:(1)We accurately targeted the C-terminal loop was responsible for the instability of PGUS by combining B-factor and RMSF analysis,and designed several C-terminal loop-truncated mutants.The thermostability of D590-604 and D591-504 was increased by 1.5 times at 70?,and the Gibbs free energy of unfolding(?G)was increased by6.8 k J mol^-1 compared with PGUS(31.9 k J mol^-1).In the meanwhile,the expression of the mutant was also increased by 77.1%and the catalytic efficiency k_cat/K_m was increased by 1.6-fold.In addition,we found that the C-terminal loop deletion reshaped the conformation of the active pocket by molecular dynamics simulations of PGUS and the substrate p NPG,and narrowed the distance between the catalytic amino acids and the substrate,which promoted the improvement of the catalytic efficiency.We established a fed-batch reaction process to achieve the efficient conversion of GL with the PGUS mutants,the GL conversion rates of D590-604 and D591-504 reached 90.5%and 84.7%,respectively,in addition,the corresponding GA yield was 91.9%and 97.4%,respectively in three times feeding,however,the wild-type PGUS was only fed one time,and GL conversion only reached 66%,the GA yield was 57.1%.(2)We found that the C-terminal loop also plays an important regulatory role in the expression and activity of GH2 family EGUS(derived from Escherichia coli),and the whole deletion of C-terminal region of EGUS(D588-603)resulted in 39.8%decrease in total expression level and 49%decrease in specific activity compared with wild-type,Nonetheless,the deletion of partial C-terminal region(D597-603)increased the soluble expression level by 63.5%without loss in specific activity.The results showed that the partial C-terminal region(residue 588-596)is necessary for maintaining the expression level and activity of EGUS.It has been reported that the entire C-terminal loop of GH2 HGUS(derived from Homo sapiens)plays an important role in the expression,activity and phosphorylation of HGUS.Based on these results,we proposed the evolutionary process of the C-terminal loop of GH2 GUSs.(3)Floop(residues 362-377)and loop470(residues 369-473)were determined as the key components of PGUS substrate channel by the analysis of 200 ns molecular dynamics simulations of PGUS and GL composite structures.The Floop played important role in recognition of the substrate GL aglycone and was responsible for the turnover of GL aglycone during catalysis;whereas loop470 was primarily responsible for the recognition of glycone,which guided the conformational and positional adjustments of GL through the dynamic movement during the catalytic hydrolysis.Combined with the crystal structure of PGUS and GAMG complexes obtained in previous studies,we elucidated the molecular mechanism of PGUS in continuous hydrolysis of GL and GAMG to GA.Based on the substrate recognition properties of PGUS,the activity of EGUS in hydrolyzing GAMG was increased by 4.6-fold by the important loop replacement.