Study On The Binding Mechanism Of Fusarium Solani Cutinase On Aliphatic Polyester Substrate

Most of the solid phase macromolecular degrading enzymes in nature have substrate binding domains,while the cutinase discovered in recent years that has degradation effects on various crystalline synthetic polymer polyesters is a single domain enzyme with only a single catalytic domain.How does cutinase accurately adsorb on the surface of polyester substrates without the assistance of binding domains,and how does it bind to different molecular chains?No clear mechanism has been reported currently.In this paper,the typical cutinase FsC from Fusarium solani was selected as the research object,and two kinds of aliphatic polyester PBS and PCL were selected as substrates.The adsorption and binding mechanism of FsC with insoluble solid polyester substrates PBS and PCL was studied by one-step site directed mutagenesis and molecular docking;Three fusion proteins were obtained through fusion PCR technology,and the effect of adding substrate binding modules on the adsorption and binding capacity of FsC was investigated,laying a theoretical and practical foundation for elucidating the mechanism of cutinase polyester degradation and efficient modification.The specific research results are as follows:（1）Study on the adsorption of solid substrates by FsCBased on the structural analysis of FsC,it is speculated that the hydrophobic amino acids on the three loop rings around the active center are involved in the adsorption of solid substrate by the enzyme.Through site directed mutagenesis and adsorption rate detection,it was determined that Loop II and Loop III played the main adsorption function.The 81st position leucine and 87th position proline on Loop II,as well as the 185th,186th,and 190th positions alanine and 189th position leucine on Loop III play a role in the enzyme adsorption process.Based on these research results,it is determined that the planes of L81,P87,A185,A186,L189,and A190 constitute the key regions for the adsorption of enzymes and aliphatic polyesters PBS and PCL.It is speculated that the flexible loop region may undergo moderate deflection during the adsorption process,making these amino acids face the surface of the polyester,better completing the adsorption of solid substrates.At the same time,we conducted a sequence and structure comparison analysis of 13polyester-degrading cutinases in the PDB database.The results showed that the loop region around the enzyme active center is a common region for all cutinases,and the loop region contains a high content of hydrophobic amino acids,further proving that the hydrophobic amino acids on the loop are a necessary condition for cutinases to adsorb solid substrates.Most cutinases contain three surface loop rings,which may achieve their adsorption of solid substrates through the hydrophobic interaction of hydrophobic amino acids on Loop II and Loop III,which are consistent with FsC;Unlike typical cutinase enzymes,Is PETase and Pa PETase,which have PET hydrolysis activity,may have hydrophobic amino acids on Loop I and Loop II to complete the enzyme’s adsorption function on the substrate.（2）Study on the binding of FsC active centers to polyester molecular chainsMolecular docking of FsC with PBS and PCL substrates was carried out.Based on the docking prediction results,eight mutants were constructed,and the kinetic parameters of FsC mutants against p-NP esters and aliphatic polyester（PBS and PCL）substrates were measured.The results showed that the catalytic ability of FsC to long-chain substrates decreased after mutation,with a preference for degradation of short chain substrates;For different substrates,FsC may rely on different forces to exert binding effects.For short chain p-NP ester substrates,when some amino acids around the active center of FsC mutate to Ala,Km decreases,indicating that the smaller the steric hindrance,the more advantageous the enzyme binds to the substrate;At the same time,the Kcat/Km of L81V is higher than that of L81A,indicating that the hydrophobic environment around the active site is also conducive to the enzymatic hydrolysis of small molecule short chain substrates.For medium long chain p-NP ester substrates,the Km values of L81A and L81V decreased,indicating that the steric hindrance at the 81st position of leucine plays a major role in the binding process between FsC and medium long chain substrates,and the steric hindrance at the positions of L51 and V184 also has a certain impact.For long-chain aliphatic polyesters,S42,which forms the oxygen hole structure,plays a crucial role in the process of enzyme binding to the substrate by forming a hydrogen bond with the substrate and stabilizing the conformation of the intermediate transition state;When using PBS and PCL as substrates,the Km value of V184A decreased,indicating that removing the gatekeeper structure at V184 is more beneficial for the enzyme to bind to long-chain PBS and PCL substrates;The role of leucine at position 189 in binding FsC to different long-chain substrates varies,possibly due to hydrogen bonding between substrates;The size of steric hindrance and the hydrophobic environment around the active site can jointly play a role in the enzyme catalysis process,but compared with short chain substrates,steric hindrance and hydrophobic interaction have no significant impact on the binding of long chain substrates.（3）Study on the effect of structural domain fusion on the adsorption and binding of FsCThe fusion proteins FsC-Linker-SBD₁SBD₂,FsC-Linker-PBM and FsC-Linker-HFB₄were successfully constructed by fusion PCR by connecting three substrate binding modules from different sources to the carboxy-terminal of FsC.And their adsorption rates on solid substrates of PBS and PCL were measured respectively.The ability of fusion protein to bind and degrade PBS and PCL substrates was tested.The results showed that the adsorption capacity of FsC to PBS and PCL solid substrate did not improve after the addition of three domains,which may be related to the source of the domain or the location of the connection.Kinetic studies showed that the Km values of FsC-Linker-PBM and FsC-Linker-SBD₁SBD₂decreased on PBS and PCL substrates,suggesting that PBM and SBD₁SBD₂ played an auxiliary role in the binding process of FsC with PBS and PCL molecular chains.The results of degrading film experiment showed that the degradation efficiency of PBS and PCL of aliphatic polyester was improved after adding PBM from Alcaligenes faecalis,which indicated that adding appropriate domain could improve the degradation ability of FsC to aliphatic polyester.This provides new information for protein modification of polyester degrading enzyme.