The Allosteric Regulation Mechanism Of Rhizomucor Miehei Lipase Via Modification Of The Propeptide

Propeptides of many proteins function as intramolecular chaperone required for the folding and correct secretion of protein,and ultimately they are proteolytically removed and degraded.When the propeptide changes,the same peptide chain of enzyme can be folded into a conformation with different spatial structures,substrate specificities,and other catalytic properties,which looks like a new enzyme.Rhizomucor miehei lipase(RML)is an important industrial enzyme.Its propeptide locates between signal peptide and functional domain.Because the mechanism of allosteric regulation of the RML via modification of propeptide is not clear,propeptide cannot be modified through rational design to efficiently improve the performance of RML.The purpose of this research is to study the function of the propeptide by mutation and fragment deletion and the regulatory mechanism of the RML structure via modification of the RML propeptide,and to obtain a good-performance novel mutant protein.Firstly,two new kinds of lipases from RML were constructed by replacing the propeptide region with that from Rhizopus chinensis lipase(RCL)and Rhizopus oryzae lipase(ROL),the sequence of which have a certain homology with gene of RML propeptide gene.Two propeptide genes of RCL and ROL were respectively ligated to the RML mature-lipase gene by overlap extension PCR.The mutant plasmids of pPIC9K-proRML were constructed and then transformed into P.pastoris GS115 cells.The new lipases with new propeptide regions were successfully expressed.The results show that the changes in the RML propeptide caused the same mature polypeptide to fold into different conformations,each with distinct characters.The activity of RCLpro-RML and ROLpro-RML was 3.4 times and 2.0 times higher than that of wild-type RML,respectively,but the stability of the new RMLs was reduced.Changes in propeptides maybe increased the overall flexibility of the spatial structure of RCLpro-RML and ROLpro-RML.Wespeculate that the conserved residues in propeptide determine the formation of the spatial structure of the RMLs,while changes in other amino acid residues of propeptide make the enzyme exhibit a variety of catalytic properties.Secondly,mutation studies on propeptide were performed.The mutant library for RML propeptide was constructed using error-prone PCR and mutation in Ser8,Pro35,and Pro47 were obtained.Seven mutant RMLs with changes in structure,activity,and stability were obtained using the combined mutations in the above obtained genetic locus.Among them,the activity,thermal stability and methanol stability of recombinase AB were better than wild-type RML.The study found that the effect of mutations in propeptide on RML may be related to the presence of mutation in conserved regions and hydrophilicity of the mutant amino acid residues.In addition,nine novel high-activity RML recombinases were obtained by saturation mutation of the above mutant amino acid sites.It was showed that at least one residue of novel RMLs was replaced by more hydrophilic amino acids.This result verified that the enhancement of hydrophilicity of the residues facilitated the interaction between the RML propeptide and the mature peptide,and had a positive effect on the enzyme activity.Thirdly,based on the analysis results of the conservation,possible structure,and hydrophilicity of amino acid residues in propeptide,RML propeptide was divided into four segments: ?(1-11AA),?(12-31AA),?(32-47AA),?(48-71AA).Through the deletion of propeptide fragment,it was found that the ?-? segment of propeptide was the crucial segment and it could independently assist RML mature peptide in folding and forming an active configuration.In the process of mature peptide folding,III and IV segments work together.The impact of ?-?segment on RML enzyme activity is minimal,but it is critical to the thermal stability of RML.The ?-? segment was subdivided into three segments E,F,and G.These three segments complement each other when they act as intramolecular molecular chaperones.We infer that,the highly conserved amino acids on ?-? segment interact with residues on mature peptides to form the basic?/? folding structure of RML lipase,guaranteeing the formation of biologically active structures,while other residues on propeptide play a role in stabilizing the structure of propeptide itself,and finely regulatingthe spatial structure of RML,and the role of ?-?segment may be to modulate the rigidity and flexibility of the RML configuration.Finally,structural models of RML propeptide and PRO-RML complex were established by using dynamic simulation techniques and molecular docking techniques,the key amino acid sites in the interaction interface between mature peptide and propeptide were analyzed.And models of mutant pro-RMLs obtained in the previous work were established by homology modeling.Based on analysis of the changes in the structure of each model and performance analysis results of related mutant RMLs,the allosteric regulation mechanism of RML via modification of the RML propeptide was obtained.During folding of RML,the propeptide interacts with mature peptide,regulates the secondary structure of RML,changes the structure and size of active center region and the solvent accessibility of the molecular surface,thus changing activity and stability of RML.After above experiments and analysis,we cleared the key amino acid residue positions and function of each fragment of the RML propeptide,and initially clarified the allosteric regulation mechanism of RML via modification of the propeptide.Guided by these mechanisms,it is possible to achieve a purposeful,rapid,and efficient modification of RML based on propeptide and to get a new type of mutant enzyme with higher application value.In addition,this study also provides a degree of reference for the modification of other lipases based on propeptide.