Study On Molecular Design Of MAS1 And PCL Lipases Based On Structural Information And Biological Computation

The discovery of lipase can be traced back to 100 years ago.As one of the first enzymes identified,lipases have now been utilized comprehensively in food,pharmaceuticals and energy industries,and using its regio-specificity to modify oils to produce high value-added structural lipids as well as its ability for mediating chemoenzymatic oxidation to prepare epoxides and lactones are two promising applications of lipases with great economic effects.However,the catalytic performances of native lipases couldn't fully meet the needs of environmentally conscious manufacturing.In the absence of an appropriate rational design strategy,enzymes engineering are mostly focused on tedious probabilistic directed evolution approaches.Therefore,in order to avoid the complicated screening process,the corresponding molecular mechanism should be resolved to guide the rational design of lipase.In the post-genome era,the discovery of molecular mechanisms could also promote the mining efficiency of new enzymes with specific properties.In this study,the biological computations,molecular biology techniques and biochemical characterization were used to investigate the molecular mechanism of regioselectiity and inactivation mechanism driven by H₂O₂ using two lipases respectively from marine Streptomyces sp strain W007 and Penicillium camembertii,called MAS1 and PCL,as the research template.The engineering strategy for regulating regioselectivity and improving H₂O₂ resistance were also propose and verified.It provides new ideas for the mechanism research and design of enzymes.The specific research contents and conclusions are as follows:1. The molecular mechanism of non-regiospecificity of MAS1 lipase and the strategy for engineering its regiospecificity?.Determining the structural basis for the regio-selectivity of MAS1 lipase.The structural alignment of MAS1 lipase showed that the catalytic pocket could be divided into three sub-regions,including Pocket A,Pocket B and Pocket C,used for accommodating three acyl chains of triglycerides,respectively.The complex structures of fatty acyls in sn-1,sn-2 and sn-3 positions of tricaprylin?TC8?bound to MAS1 lipase were obtained by homologous modeling and manual docking.The comparison of the three binding conformations of the TC8 demonstrated that the glycerol backbone of the TC8 in the sn-2 reaction mode was tightly surrounded by the pocket,limiting the space necessary for conformation adjustment.In contrast,the glycerol backbone of the TC8 in the sn-1 or sn-3 reaction mode was less restrained and the conformation of the substrate could change more freely.When the pocket bottom residue H108 was substituted with tryptophan,the MAS1 lipase no longer accommodated the TC8 in the sn-2 reaction mode,and lost the sn-2 site activity of TAG.It should be noted that the mutant H108W could convert into a sn-1,3regiospecific lipase,as the mutation might not affect the binding of the TC8 in the sn-1 or sn-3 reaction mode.Moreover,the molecular dynamics?MD?simulation and mutation experiments of catalytic residues S109 and H232 revealed that a binding site composed of H108,G37 and N45 in the Pocket B of MAS1 lipase was observed for stabilizing the non-reactive sn-1 moiety of the TC8 in sn-2 reaction mode.The presence of the site could improve the stability of substrate in sn-2 reaction mode.This characteristic also contributed to the sn-2 site activity and non-regiospecificity of MAS1 lipase.Thus,reshaping the site may achieve the modification of regioselectivity of the lipase.?.The engineering strategy for the regiospecificity of MAS1 lipas.The range of PSI value of the mutants with mutation at G40,N45,H108 and T237 located in Pocket B was from-30 to 100.It showed that the regioselectivity of the lipase can be designed by protein engineering.The regulation for regioselectivity of MAS1 lipase was summarized from the MD simulations.Increasing the electrostatic interaction between Pocket B and the polar part?glycerol backbone?of TC8 binding in sn-2reaction mode,the absence of the group with negative charge at the binding site for non-reactive carbonyl and enhancing the conformation stability of Pocket B by interaction all could improve the sn-2 regio-preference.And introducing residues with bulky side chain into Pocket B to seal the entrance of substrate in sn-2 reaction mode into pocket or block the binding site of the non-reactive carbonyl group of TC8 with same conformation,however,could reduce the sn-2 region-preference of the lipase and even transformed it into a sn-1,3 regiospecific lipase.2. The detailed inactivation mechanism of PCL driven by H₂O₂and the strategy for improving its H₂O₂-resistance?.The inactivation mechanism of PCL driven by H₂O₂.The H₂O₂ was bound to PCL by forming hydrogen bonds with the residues Y21,H144 and H259.The result of CMD simulation showed the polar part of PCL's catalytic pocket accommodated 2to 4 H₂O₂ molecules.The SMD simulation revealed that H₂O₂ molecule entering from solvent to its binding site was thermodynamically and kinetically favorable.In other word H₂O₂ preferred to stay at the region.Meanwhile,QM/MM MD simulation combined with mutation experiments proved that the changed chemical properties of oxidation-sensitive residues Y21 and H144 would lead to an impaired activity of PCL.?.The engineering strategy for improving the H₂O₂ resistance of PCL.In order to improve the H₂O₂ resistance of PCL,we proposed to introduce polar amino acids at the top of polar pocket.The formed bonds between introduced amino acids and H₂O₂would afford a higher energy barrier for H₂O₂ entering the catalytic pocket.CMD simulation results showed that the mutants Y84R,F256Y,I260E and I260R could effectively reduce the number of H₂O₂ molecules located in the pocket.Especially,Y84R and I260R had the best effect.The half-lives of mutants F256Y,I260E,Y84R and I260R incubated with 1 M H₂O₂ concentration were 1.41,3.66,4.17and 4.40 times higher than that of the wild-type,respectively.This observation was consistent with the theoretical prediction.The SMD simulation results showed that mutants Y84R and I260R imposed a very significantly higher energy barrier for H₂O₂to access the active sites by the hydrogen bond between introduced arginine and H₂O₂.But the tendency of H₂O₂ getting access to the polar pocket did not change.QM/MM MD simulation revealed that the free energy characteristics of perhydrolysis reaction catalyzed by mutant I260R was closed to PCL-wt,while mutant Y84R increased the energy barrier of the rate determining step.Therefore,a parallel catalytic efficiency was observed between I260R and the wild-type.Noteworthy,the catalytic efficiency mutant Y84R decreased significantly.The results were consistent with the biochemical experiments.Similar results were obtained using the above strategy with regard to other two homologous lipases.Therefore,the strategy could be generalized in enzymes related to H₂O₂.