Studies On The Biochemical Properties And Applications Of Four Enzymes Involved In Bacterial Polysaccharide Biosynthesis

Like nucleic acids and proteins,glycans in the form of oligosaccharides and glycoconjugates(glycoproteins and glycolipids) are vital biopolymers found in organisms across all domains of life.They play critical roles in mediation of numerous complex biological processes.Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation,illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.Important glycan structrues in human body include tumor-associated antigens and ABH blood group antigens.Altered expression of glycans constitutes a hallmark of the tumor phenotype.Several glycan structures are commonly found on malignant cells.They include Tn antigen,T antigen,sialyl Lewis x,Lewis y,gangaliosides,Globo-H and polysialic acid.These tumor-associated glycan structures can serve as diagnostic markers for cell malignancy and have also been exploited as targets for cancer immuno-therapy via the development of glycan-based vaccines.ABH system is one of the most common and important blood group systems in transfusion medicine.The structures of the ABH glycan epitopes are defined as GalNAc-α1,3(Fuc-α1.2)-Gal(A antigen),Gal-α1.3(Fuc-α1.2)-Gal (B antigen) and Fuc-α1,2-Gal(H antigen).Available of these oligosaccharides are required for their functional study and therapeutical applications.The bacterial cell surface is decorated with remarkable variations of polysaccharide (PS) structures.One major type of them,O-PS,is a major component of the bacterial cell surface lipopolysaccharide(LPS).It is composed of multiple copies(as many as 100) of an oligosaccharide repeating unit(O-unit).O-PS plays an important role in mediating bacteria-host interactions such as the effective colonization of the host and the resistance to complement-mediated immune responses.The illumination of their synthesis pathway would assist the medical application related to baterial deseases.The biosynthesis of saccharides in nature uses glycosyltransferase.They transfer a given monosaccharide from the corresponding sugar nucleotide(sugar donor) to a specific hydroxyl group of a sugar acceptor.With obvious advantages of achieving high regio- and stereochemistry of glycosidic bonds,often in a high efficient manner,glycosyltransferasecatalyzed enzymatic synthesis of saccharides becomes an attractive and powerful alternative to the chemical synthesis.Compared with their mammalian counterparts, bacterial glycosyltransferases are more easily overexpressed as soluble and active forms without complicated gene manipulation techniques.Furthermore,they appear to have broader substrate specificity,thereby offering tremendous advantages in the enzymatic synthesis of oligosaccharides and their analogs.In addition,various bacteria exhibit structural mimicry of mammalian glycans on cell surfaces.Therefore,corresponding glycosyltransferases can be explored for the synthesis of human-like glycans.One the aim of this thesis is exploring bacterial glycosyltransferases for enzymatic synthesis of oligosaccharides.We have identified and charactered three glycosyltransferases from E.coli O-antigen biosynthesis gene cluster.In addition to the biochemical study towards the illumination of their substrate adaptability,enzymatic dynamics and catalytic mechanism,the synthetic applications of these glycosyltransferases were also elucidated by the synthesis of fucosylated or lacto-series oligosaccharides in small scale.Fucosylated carbohydrate structures are involved in a variety of biological and pathological processes in eukaryotic organisms including tissue development,angiogenesis, fertilization,cell adhesion,inflammation,and tumor metastasis.In contrast,fucosylation appears less common in prokaryotic organisms and has been suggested to be involved in molecular mimicry,adhesion,colonization,and modulating the host immune response. Fucosyltransferases(FucTs),present in both eukaryotic and prokaryotic organisms,are the enzymes responsible for the catalysis of fucose transfer from donor guanosine-diphosphate fucose(GDP-Fuc) to various acceptor molecules including oligosaccharides,glycoproteins, and glycolipids.We have identified and characterized two bacterialα1,2-FucTs and synthesized fucosylated oligosaccharides with the recombinant enzyme.The wbsJ gene from Escherichia coli O128:B12 encodes anα1,2-FucT responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via anα1,2 linkage.The wbsJ gene was overexpressed in E.coli BL21 as a fusion protein with glutathione S-transferase(GST) at its N-terminus.GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galβ1,3GalNAc(T antigen), Galβ1,4Man and lactose being better acceptors than Galβ-O-Me and galactose.Lactulose (Galβ1,4Fru),a natural sugar,was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose.Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with K_m values of 7.81 mM and 13.26 mM,respectively.However,the k_cat/K_m value of lactose(6.36 M^-1·min^-1) is two times lower than that of lactulose(13.39 M^-1·min^-1).In addition,theα1,2-FucT activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn²⁺ or Mg²⁺.This activity was competitively inhibited by GDP with a K_i value of 1.41 mM.Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H¹⁵²xR¹⁵⁴R¹⁵⁵xD¹⁵⁷.In contrast toα1,6-FucTs, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However,residues R¹⁵⁴ and D¹⁵⁷ were found to play critical roles in donor binding and enzyme activity.H¹⁵² and R¹⁵⁵ are also important in GDP-Fuc binding.The results suggest that the common motif shared by bothα1,2-FucTs andα1,6-FucTs may have different functions.Enzymatic synthesis of fucosylated sugars in mg-scale was successfully performed using Galβ-O-Me and lactoseβ-N₃ as acceptors.Anotherα1,2-FucT encoding gene,wbwK,from E.coli O86 was also expressed and purified as a GST fusion protein.It has similar features with WbsJ regarding metal ion independence.But WbwK shows strict substrate specificity and only recognizes Galβ1,3GalNAcoα-OR(T antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen.In contrast to otherα1,2-FucTs,WbwK does not display activity toward the simple substrate Galβ-OMe.WbwK and WbsJ share a common acceptor substrate,Galβ1,3GalNAcα-OR,but WbsJ has broad acceptor specificity.A hypothesis has been proposed that the high variable regions in glycosyltransferase determined acceptor specificity.To verify this hypothesis,a swapping experiment between variable regions of WbsJ and WbwK has been performed leading to six chimeric enzymes.Even though region swapping resulted in activity loss of three chimeras,other three chimeras showed decreased activity but broad acceptor specificity,indicating the possible roles of the variable regions for acceptor adaptability determination.The glycosyltransferase-catalyzed glycosylation reactions require high-energy sugar nucleotides as donor substrate.Specifically,for FucT,GDP-Fuc is needed.GDP-Fuc can be generated from GDP-mannose via de novo pathway or from fucose,ATP and GTP via salvage pathway.We overexpressed an enzyme(FKP) from bacterium Bacteroides fragilis responsible for the only identified GDP-Fuc salvage biosynthesis pathway in prokaryote.In addition to its application potential for feasible production of GDP-Fuc indicated by in vitro reaction,FKP also showed promiscuity for different fucose analogs through CE analysis of FKP reaction with nine fucose analogs as substrate.To investigate its promiscuity in vivo, we introduced this enzyme into E.coli O86:B7 strain of which the GDP-Fuc de novo pathway was disrupted.The matant strain with engineered GDP-Fuc biosynthesis pathway was feeded with a panel of unnatural fucose analogs.These analogs were successfully incorporated into bacterial surface polysaccharides to generate structurally novel polysaccharides.Selective chemical reactions were carried out in vitro to further elaborate the functional groups(azido and amino groups) appended to the polysaccharides.In conclusion,we provide a general,facile and effective means to introduce modifications into polysaccharides in vivo.The technology presents a powerful tool to dissect the functions and roles of polysaccharide in biological processes.Galactose is commonly found in several classes of glycoconjugates in both prokaryotes and eukaryotes.Various galactosyltransferase(GalT) enzymes catalyze the addition of galactose(Gal) in two anomeric configurations throughα1,2-,α1,3-,α1,4-,α1,6- orβ1,3-,β1,4-linkages.The variety of galactosylation reactions significantly contributes to the tremendous diversity of oligosaccharide structures expressed by living organisms.Lacto-N-biose I disaccharide(Galβ1,3GlcNAc),also known as type 1 chain,is the precursor of a number of important carbohydrate epitopes in human body,such as Lewis a,Lewis b and sialyl Lewis a antigens.Galβ1,3GlcNAc motif is also present in other important oligosaccharides,such as lacto-N-tetraose(LNT).Aβ1,3-GalT(WbgO) was identified from E.coli O55:H7.It was overexpressed with a GST fused at its N-terminal and purified by GST affinity chromatography with a specific activity of 67.5 mU mg^-1.Itsβ1,3-GalT activity was verified by radioactive assay and structure characterization of the synthesized disaccharide by ESl-MS and NMR.Fusion with GST is essential for its soluble expression and efficient purification.The fused enzyme catalyzed galactosyl-transferring reaction under pH 6.0-8.0 with an optimal pH of 7.0.It also showed preferce for different pH system with HEPES buffer as the best among the four buffer systems which have been used in the assay.Its activity is dependent on certain divalent metal ions(Mn²⁺ or Mg²⁺).N-acetylglucosamine(GlcNAc) and oligosaccharide with GlcNAc at the non-reducing end were shown to be its predominant acceptors. However,N-acetylgalactosamine(GalNAc) and oligosaccharides with GalNAc at the non-reducing end were also accepted.Lacto-N-triose appeared to be the best acceptor among the tested acceptors.The kinetic parameters for the donor substrate and three acceptors were estimated,which are consistent with the substrate specificity study.At last, LNT was synthesized with the purified GST-WbgO using lacto-N-triose as the sugar acceptor.The product structure was confirmed by ESI-MS and NMR analysis.The synthetic reaction resulted in a complete convertion of the sugar acceptor,indicating the potential usage of this enzyme for oligosaccharide synthesis.In conclusion,focusing on the biochemical characterization of three glycosyltransferases and one sugar nucleotide synthetase,studies in this thesis enrich the basic knowledge of the enzymes related to bacterial polysaccharide synthesis.Furthermore, feasible synthesis methods for fucosylated oligosaccharide and lacto-N-tetraose and a novel bacterial polysaccharide remodeling technology have been developed based on these enzymes.