Experimental And Theoretical Research On The Effect Of Salt Bridge On The Thermal Stability Of Clostridium Absonum 7?-Hydroxysteroid Dehydrogenase

Clostridium absonum 7?- hydroxysteroid dehydrogenase(CA 7?-HSDH) mediates the converting process from TCDCA to TUDCA, TUDCA is the endemic and the main active ingredient of bear acid, the rare Chinese animal medicine, so this enzyme not only have great application prospects and economic value in the biotransformation and industrial production but also a great significance in protecting bears and mainteing ecological balance. Previous studies in our lab have resolved the crystal structure of CA 7?-HSDH, the result shows the molecule contains a lot of salt bridges, many scholars hold different attitudes to whether the salt bridges contribute to the thermal stability of the protein or not, many of the studies on the thermal stability of the enzyme-related factors indicated that the way of salt bridges effect heat resistance of protein were mixed, so the research on the impact of salt bridges for CA 7?-HSDH's thermal stability is an important prerequisite for improving the enzyme's thermal stability. This issue take CA 7?-HSDH as the object for the study, using MD simulations find stable salt bridges and remove them, detecting the enzyme function and observing the effect of the salt bridges for thermal stability of CA 7?-HSDH.This study includeds site-directed mutagenesis experiments and molecular dynamics simulations: discuss the effect of salt bridges for 7?-HSDH's thermal stability from site-directed mutagenesis experiments, and analys the causes from the perspective of molecular dynamics simulation.(1) Through analysing the trajectory of 300 K MD simulation to find the stable salt bridges which their O-N distance fluctuation range within 3.5?. There are 4 pairs of salt bridges can be designed to salt bridges destroyed site-directed mutagenesis, we analyzed the thermal stability related enzyme function of purified wild-type enzyme and the mutant enzyme. The results show the D49 N mutant is inactivated, optimum temperature of E24 Q, D85 N and D206 N decreased from 35?(WT) to 25?, and WT still had about 20% of the relative enzyme activity after incubation under 45 ? for 2 hours, but each mutant under the same conditions have been deactivated. Half-life tests show at 15? and 25? each enzyme's t1/2 changed little, but at 35 ? WT's t1/2 is 0.39 h, every mutant enzymes' t1/2 increased various, when at 45? the WT's t1/2 is 0.13 h, each mutant enzymes' half-life could not be detected. Tm value test results reveal that Tm value of the mutant enzymes decreased from WT of about 72 ? to 65 ?. Experimental results show that the thermal stability of the mutant enzymes is descended.(2) Using Amber force field to do 5ns MD simulation on wild-type and mutant enzymes under 300 K, 310 K and 320 K conditions. The RMSD values of protein backbone show that only D206 N under 320 K undergone significant increase. The RMSF values show similar flexibility under 300 K among all the enzymes but the D49 N, we presume the D49N's inactivation in experiments because of the overall improvement of the flexibility; the flexibility of E24 Q, D85 N and D206N's catalytic triad under 310 K are slightly higher than the WT's, under the conditions of 320 K each mutation system have undergone greater flexibility increased as a whole, indicating that under 310 K, the rigidity of the residue sequence associated with the protein structure has been maintained, but its active site-related residues' flexibility increased moderately in order to adapt distorted substrate and coenzyme molecules in thermal environment. We suggesting that the rigidity's maintain and the flexibility's appropriatly increase is the reason for mutant enzymes' t1/2 is longer than that of WT's at 35? environment. The flexible structure of the region where the amino acids at the catalytic center must ensure that within a certain range, below or above this range will result in inactivation of the protein, and amino acids in other rigion maintain relative rigid can help maintaining structure stability of protein. Structure under 320 K analysis showed obvious structural changes in the D206N's C-terminal; 320 K polar contact analysis showed that polar interactions between mutants' relative amino occur reduction; and the salt bridge between Asp206 and Arg262 and their located C-terminal region play an important role of maintaining structural stability.