A Study On The Enzymatic Properties Of Gibberella Zeae Lipase GZEL And Function Of C-terminal Peptides On Its Catalytic And Interfacial Adsorption Properties

Gibberella zeae is the sexual stage of Fusarium graminearum.Gibberella zeae is the causative agent of Fusarium Head Blight?FHB?,one of the most destructive plant disease of cereals,accounting for high grain yield losses.Gibberella zeae lipase?GZEL?is one of extracellular lipases secreted by G.zeae,which was reported to account for its pathogenicity.Presently,the crystal structure of GZEL has been resolved,the biochemical properties of this enzyme was still unknown.Furthermore,the difference between GZEL and other reported lipase is that GZEL owns unique C-terminal structure according to the three-dimensional structure of the protein,while the function of C-terminal peptide has not yet been elucidated in detail.An in-depth understanding of the enzymatic properties of GZEL and the function of the C-terminus on enzyme activity not only helps to understand the structure-function relationship of the enzyme,but also lays the foundation for the future molecular transformation and industrial application of lipase.What's more,it provides guidance to prevent and control diseases caused by Fusarium graminearum.In the present study,we obtained recombinant GZEL and conducted a comprehensive study of enzymatic properties of GZEL using the emulsified and monomolecular film technique.Moreover,we designed mutants deleting C-terminal peptide and analysed the effects of C-terminal peptide on substrate selectivity and interface adsorption of GZEL.Furthermore,molecular docking simulations and structural analysis were combinated to explore the structural basis of behavior of GZEL.The results are as follows:?1?Biochemical characterization of the lipase GZEL based on the classical emulsified system.We constructed the recombinant E.coli ShuffleT7 containing the gene of lipase GZEL.The target protein was purified by affinity chromatography and ion exchange chromatography.Both phospholipase and glycolipid hydrolytic activities were observed,except for the predominant lipase activity,and the optimum reaction conditions were as follows:temperature was 30-40°C,pH was 7.0?lipase activity?;temperature was 45°C,pH 6.0-7.0?phospholipase activity?;temperature 50°C,pH 6.0?glycolipid hydrolytic activity?.The kinetic parameters showed that the order of substrate affinity of GZEL was triglyceride>sucrose ester>phospholipid.GZEL showed high stability at 50°C,the t_1/2 value was 37.6 min at 60°C.GZEL preferred medium chain-length triglycerides.?2?Substrate selectivity of GZEL was further studied using monomolecular film technique,which is more sensitive and easier to control compared with emulsified system.The preference order of GZEL to different phospholipids was PS>PG>PC>PI>CL>PA>PE,while no hydrolytic activity was detected for SM.Moreover,GZEL showed higher galactolipase activity on 1,2-distearoyimonoglacto-sylglyceride?MGDG?,indicated that GZEL could potentially recover polyunsaturated fatty acids on galactolipids.A kinetic study on the stereo-and regioselectivity of rGZEL was also performed by using three pairs of pseudodiglyceride enantiomers?DDGs?.GZEL presented higher preference for distal DDG enantiomers than adjacent ester groups,however,no hydrolytic activity to sn-2 position of diglyceride analogs was found.Furthermore,GZEL preferred R configuration of DDG enantiomers.Molecular docking of DiC8-PS and DiC8-MGDG with GZEL revealed that they both interacted strongly with the catalytic pocket of GZEL,which is consistent with the results that GZEL exhibit phospholipase activity and galactolipid activity.Docking of 1,3-DDG?S2?and 1,3-DDG?R2?with GZEL indicated that R2 bind more closely to the catalytic pocket and promote catalytic reaction,confirming GZEL preferred R configuration.?3?Effect of C-terminal peptide on the substrate selectivity of GZEL.C-terminus of GZEL is composed by two secondary structure segments,with a loop and a?-helix next to it.Truncated mutants?GZEL-???-helix?and GZEL-???-helix+loop??were designed and successfully expressed in E.coli BL21 Star?DE3?plysS.The reagent sarcosine was used to promote dissolution while the protein is inclusion body.After nickel column and DEAE column purification,we obtained the high purity proteins of the mutant.Both the lipase activity and the phospholipase activity of the mutants were reduced,and the C-terminal peptide had greater effect on lipase activity than phospholipase activity,furthermore,The?-helix segment plays a more important role on lipase activity exhibition of GZEL.Compare to GZEL-wt,hydrolytic activities of GZEL truncated mutants to all the anionic phospholipids?PS,PG,CL,PI,PA?were significantly decreased,however,the C-terminal peptide had little influence on hydrolysis activities to zwitterionic phospholipids?PC and PE?.The hydrolytic activities of the mutants on MGDG were both reduced.?4?Function of C-terminal peptides on interfacial adsorption properties of GZEL.Catalytically active serine mutants were designed to avoid the influence of hydrolysis on the adsorption.The maximum insertion pressure?MIP?of GZEL and its mutants to PE was the highest,suggests that monolayer binding of the protein is more favored in the presence of PE.All proteins investigated showed synergy factor a>0,corresponds to the positive interaction between proteins and substrates.??₀ of mutants were greater than equilibrium surface pressure,demonstrating that proteins prefer to migrate from water subphase onto interface and bind to phospholipids.Binding kinetic parameters k_a,k_d,K_adsds showed that?-helix structure on the C-terminal of GZEL has great influence on the binding of enzyme to the phospholipids.Rich in charged amino acids in loop segment?especially for Arginine?and the amphiphilic structure of?-helix could be the structural foundation that favor the GZEL binding and interaction with the monolayer,and electrostatic interactions were involved in the binding of several protein and peptides onto membranes.The interfacial binding function of?-helix structure was verified by connecting it to the C-terminal of Aspergillus oryzae lipase?Mdl B?.