| Glycosaminoglycans(GAG)are a kind of biological macromolecule with complex structure,and a linear non-branching acidic polysaccharide formed by repeated disaccharide units,which is composed of hexuronic acid and hexosamine or their sulphide monosaccharides.Glycosaminoglycans are widely distributed and have various types,including glycosaminoglycans without sulfuric acid modification(hyaluronic acid)and glycosaminoglycans with sulfuric acid modification,including dermatin sulfate/chondroitin sulfate,heparan sulfate/heparan sulfate,keratin sulfate,etc.Heparin(HP),an important glycosaminoglycan,is composed of D-β-glucuronic acid(or L-α-eduronic acid)and N-acetylglucosaccharide forming the repeated disaccharide units.Heparin can enhance the activity of antithrombin Ⅲ which is a natural serine protease inhibitor,and activate all serine proteases that inhibit endogenous clotting pathways,so it has natural activity of anticoagulant.It has been clinically used as anticoagulant drugs for more than 100 years.In addition,heparin also mediates a variety of important physiological processes,including transport and adsorpt lipid,develop embryonic,remodel tissue,bacterial/viral infection,by binding to other proteins.At present,heparin used in clinical is most extract from animal tissue.Animal derived heparin is extracted from the mucosa of the small intestine of pigs or cattle.Due to the different extraction methods,the structure,molecular weight and biological activity of heparin products are different.At the same time,heparin contamination may be caused by animal susceptibility to infectious prion diseases.Non-animal source heparin is obtained by chemical or enzymatic synthesis methods.The structure of heparin is relatively complex,when chemical methods are used to prolong the sugar chain,many cases should be considered,including the protection and de-protection of unstable groupsprotection,activation and combination,as well sa regioselectivity and stereoselectivity,so the synthesis process is complex and the yield is low.In recent years,researchers have turned their attention to chemoenzymatic synthesis of heparin,which uses glycosyltransferase to synthesize the heparin sugar chain,and then catalyzes the isomerization and sulfurization of the sugar chain by isomerase and sulfotransferase.The substrate specificity of those enzymes is high,the product structure is single,and the reaction conditions are very mild,so the process is simpler,conversion rate is higher than chemical synthesis.At present,chemical enzymatic often use Escherichia coli K5 N-acetylglucosaminyltransferase(KfiA)and Pasteurella Multocida Heparosan Synthases 2(PmHS2)to synthesis the skeleton of heparin.PmHS2 is a dual-function enzyme with α-1,4-N-acetylglucosamine transferase(α-1,4-GlcNAc-T)activity and β-1,4glucuronic acid transferase(β-1,4-GlcNAc-T)activity,has been widely used in chemical enzymatic synthesis system.Until the writing of this paper,only two members of the dualfunction heparosan synthase family were found,PmHS2 and Pasteurella Multocida Heparosan synthases 1(PmHS1).In this study,more members of the dual-function heparosan synthase family were explored,the amino acid-molecular structure-catalytic activity of the enzyme were expanded,and the catalytic activity of heparin heparosan synthase was improved by protein engineering.Based on the above background,this paper mainly includes:1.Two new members of the dual-function heparosan synthases family were discovered and confirmed:Based on PmHS2.we identified two gene sequences of suspected dual-function heparosan synthases in strains Mergibacter Septicus and Psittacicella gerlachiana.which were named MsHS and PgHS,respectively.We successfully obtained the protein of MsHS and PgHS encoded by the above two genes through the recombinant expression system in Escherichia coli.Using UDP-GlcNAc,GlcA-pNP or UDP-GlcA,GlcNAc-GlcA-pNP as substrates,MsHS and PgHS glycosyltransferase activities were preliminarily determined by High Performance Liquid Chromatography(HPLC).The molecular weight and structure of the product were confirmed by Electrospray ionization mass spectrometry(ESI-MS)and Nuclear magnetic resonance(NMR).Finally,MsHS and PgHS were identified as novel dual-function heparosan synthases.2.The enzymatic properties of MsHS and PgHS were studied.The effects of temperature.pH and metal ions of reaction system on the catalytic activity of MsHS and PgHS were systematically analyzed.The kinetic constants of MsHS and PgHS were determined(Kcat and km).The results showed that the catalytic efficiency(Kcat/(kmA*kmD))of GlcNAc-T and GlcA-T activity of MsHS was higher than that of PmHS2.especially the catalytic efficiency of GlcNAc-T activity was about 7 times that of PmHS2.The catalytic efficiency of PgHS is lower than that of PmHS2.The catalytic efficiency from high to low is:MsHS>PmHS2>PgHS.which provided amino acid-activity information for the protein engineering of PmHS2.3.Extended molecular structure-catalytic property information of PmHS2:Based on the amino acid sites,activity differences and advanced protein structure prediction analysis of new members of the dual-function heparosan synthases family discovered in this paper,9 amino acid sites were selected for rational site-directed mutation of PmHS2 to obtain a series of PmHS2 mutants:By analyzing the activity of recombinant protein,it was found that the GlcNAc-T activity of the mutant which mutated isoleucine 476 to phenylalanine(PmHS2I476F),the mutant which mutanted threonine 326 to serine(PmHS2-T326S)and the mutant which mutanted phenylalanine 197 to tyrosine(PmHS2-F197Y)was 4.33,3.73 and 3.29 times that of wild type PmHS2,respectively.Meanwhile,GlcA-T activity of the mutant which mutanted threonine 344 to serine(PmHS2-T344S),the mutant which mutanted serine 130 into threonine(PmHS2-S130T)and PmHS2-F197Y was 3.35,2.97 and 2.73 times that of wild type PmHS2,respectively.Subsequently,by docking with substrate molecules through structural simulation,we found that Threonine 326(THR 326)and Threonine 344(Threonine 344)located in hypervariable region 3(HV3).When THR 344 mutated into Serine,the active pocket was significantly larger than that of wild type PmHS2.Combined with the information about HV3 binding to receptor substrates mentioned in the literature,we hypothesized that mutations in THR 326 and THR 344 affect enzyme catalytic activity by affecting active pocket size and substrate binding,and THR 326 and THR 344 were selected as the focus of the study.4.Molecular modification of PmHS2:Using the PmHS2-T344S which has higher activity as the starting protein,saturation mutation at THR 326 site was completed and two mutants whose catalytic activity increased to 1.5 times that of wild-type PmHS2 were abtained,the mutant who has serine at site 326 and 344(PmHS2-344S-326S),and the mutant who has serine at site 344 and tryptophan at site 326(PmHS2-344S-326W).Meanwhile,combined saturated mutations of THR 326 and THR 344 were carried out on PmHS2 through the high-throughput screening based on β-glucuronase which can hydrolysis GlcA-pNP.26 mutants with enhanced activity were obtained,among them,the GlcNAc-T and GlcA-T activities of the mutant who has methionine at site 326 and alanine at site 344(PmHS2-326M-344A)was about 3 times that of wild-type PmHS2;Through the analysis of the advanced structure of the mutants obtained in the saturated mutation experiment,it was found that the active pocket diameter of the mutants which have higher activity increased,which provided evidence for the previous hypothesis that the mutations of THR 326 and THR 344 affected the enzyme catalytic activity by affecting the active pocket size and substrate binding. |
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