Identification And Enzymological Characterization Of A Heparosan-alpha-1,4-GlcNAc Glycosyltransferase And Its Surface Display Expression On E.coli

Heparan sulfate(HS)is a glycosaminoglycan(GAG)composed of repeated disaccharide units,which was formed glucoside bonds between glucuronic acid(GlcA)or iduonic acid(IdoA)and glucosamine(GlcN)of N-site sulfated or acetylated.HS widely exists on the surface of mammalian cells and endothelial cells.An important characteristic that usually distinguishes HS from heparin(HP)is the degree of sulfation modification at the N-sites.HP generally has a degree of N-sulfonation higher than 70%,while the degree in HS is usually less than 50%,which is consistent with the degree of O-sulfation.HP is a macromolecular linear polysaccharide with clear biological activity.In addition to its anticoagulant activities,HP also has the characteristics of anti-inflammatory,anti-tumor,anti-atherosclerosis,anti-infection,and anti-proliferation.These activities are also mediated by the interaction between heparin and proteins.Heparosan is an unsulfurized backbone of HP,which is consistent with the structure of some bacterial capsular polysaccharides.Heparosan can be used as a precursor to synthesize animal-free derived HP with anticoagulant activity,or non-anticoagulant HP derivatives.Heparosan can usually be obtained from bacteria fermentation broth or enzymatic synthesis in vitro.The study found that some glycosyltransferases(GTs)in Escherichia.Coli O10:K5:H4(ATCC23506)and Pasteurella multocida can synthesize heparosan.With the continuous development of carbohydrate drugs,synthesis of uniform structure and identified animal-free origin of HP has become the focus of research.By simulating the biosynthesis process of HP in vivo,heparosan is synthesized by efficient and specific GTs,then isomerized and sulfated by chemical or enzymatic methods.A set of biomimetic synthesis system based on chemical enzymatic synthesis of HP was established.This synthesis method has a high yield,and can synthesize HP with definite structures.Therefore,heparosan-?-1,4-GlcNAc GTs(KfiAs)has a high application value in the synthesis of animal-free derived HP.KfiA belongs to the GT45 family of Carbohydrate-Active enZYmes Database(CAZy).But so far,the only member of the family is EcKfiA from E.coli O10:K5:H4.The insufficient number of known members limits the study of the molecular structure and catalytic mechanism of the KfiA superfamily,and limits the application of this enzyme family in the synthesis of sugar polymers.The discovery of new members of the family,the studied of catalytic mechanism and key amino acid sites not only have important theoretical significance,but also lay a foundation to remodel KfiA by protein engineering in the future to obtain better catalytic properties of HP synthases.Therefore,this paper is based on this development,including the following contents:1.A new member of the KfiA family was discovered from Gallibacterium anatis:the enzyme encoded by the G.anatis gcbd gene,and it shows a high protein primary structural homology(-58%)with the heparosan-?-1,4-GlcNAc glycosyltransferase EcKfiA derived from E.coli O10:K5:H4.The soluble recombinant expression of the enzyme(named GaKfiA)in E.coli was successfully constructed,and the protein purification method of the recombinant enzyme was established.GaKfiA uses GlcA-pNP as the acceptor substrate and UDP-GlcNAc as the monosaccharide donor.The chemical structure of the disaccharide produced by GaKfiA enzymatic reaction was systematically analyzed,and it was determined that GaKfiA showed the activity of heparosan-?-1,4-GlcNAc transferase.2.The kinetics and enzymatic properties of GaKfiA were studied,and the donor substrate specificity was analyzed.3.The crucial effect of DXD motif on the catalytic activity of KfiA family members was determined:the protein-molecular docking model was used to analyze the effect of DXD motif on the catalytic activity of KfiA family.After rational mutation,the mutant protein of GaKfiA(N56D)was obtained by recombination expression.Compared the kinetic parameters of the enzymatic reaction between GaKfiA(N56D)and GaKfiA,and the key amino acid sites of GaKfiA were identified.4.The surface display of the catalytic activity of KfiA family members was successfully realized:the recombinant protein of EcKfiA was expressed on the surface of E.coli by ice nucleus protein(INP),and the recombinant strain of EcKfiA anchored on the surface had the activity of prolonging PNP GlcA-pNP to GlcNAc-GlcA-pNP.