| Rhamnose is a common component of many bacterial polysaccharides,such as lipopolysaccharides,extracellular polysaccharides,capsular and cell wall polysaccharides.In addition,rhamnose-containing compounds are extensively present in various other organisms,including virus,fungus,plant and zootic,and play an important role in various biological processes.Studies on rhamnose and related topics have been hot since the rhamnose biosynthetic pathway was first discovered in 1960s.Accordingly,significant progresses have been made in various aspects in the fields.For example,biosynthetic pathways of rhamnose donors including deoxythymidine diphosphate-L-rhamnose(dTDP-Rha),uracil diphosphate-L-rhamnose(UDP-Rha)and guanine diphosphate-D-rhamnose(GDP-Rha)had been clarified,and activities of several rhamnosyltransferases and rhamnosidases had been demonstrated.Morover,the potentials of applying rhamnose-containing polysaccharides and other compounds to the treatment of various diseases have been demonstrated,and the enzymes involved in rhamnose biosynthetic pathway were shown to be useful drug targets.Apparently,rhamnose and related compounds and biosynthetic pathways have enormous values and an excellent platform for new drug development.However,the problem is that currently it is difficult to obtain these molecules in structurally homogeneous and defined forms.To address this problem,chemoenzymic synthesis to access rhamnose containing compounds by using rhamnosyltransferase has become a hot topic.Streptococcus pneumonia serotype 23F expreses abundant rhoamnose-containing polysachcairdes.Thus,this study is focused on the study of rhamnosyltransferase and related enzymes from S.pneumonia serotype 23F.The thesis includes four chapters:In chapter 1,studies related to rhamnose have been reviewed.Rhamnose and related topics have been attractive since the discovery that it exists extensively in bacteria but not in mammal and that rhamnose-containing compounds were valuable in tumor immunotherapy and in the treatment of antibacterial infections.As a result,studies of the functions and biosynthetic pathways of rhamnose and rahmnose-containing compounds have made significant progresses.This chapter has reviewed first the studies on the biosynthetic pathways of three rhamnose donors,dTDP-Rha,UDP-Rha and GDP-Rha,as well as as the functions and crystal structures of related enzymes.dTDP-Rha is the most common rhamnose donor,and four enzymes,included glucose-1-phosphate thymidylyltransferase RmlA,dTDP-Glc-4,6-dehydratase RmlB,dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC,and dTDP-4-keto-Rha reductase RmlD,were related to its biosynthesis.Next,several known rhamnosyltransferases from Geobacillus stearothermophilus,Saccharopolyspora spinose,Mycobacterium tuberculosis,Pseudomonas aeruginosa and S.pneumonia were reviewed.However,in these studies,gene knockout and radio labeled substrates were used to verify the functions or activities of rhamnosyltransferases,and it was almost impossible to obtain and characterize the enzymatic reaction products.Subsequently,the applications of rhamnose-containing compounds to the treatment of deseases are reviewed.In addition,progresses in the study of the biosynthetic pathways of pneumococcal capsular polysaccharides and the involved glycosyltransferases,which have great potential to become useful targets for drug development and powerful tools for chemoenzymatic oligo/polysaccharides synthesis,are also reviewed.Chapter 2 describes the development of an effective one-pot four-enzyme synthetic method for dTDP-Rha in this research.Currently,dTDP-Rha is very difficult to access,which has hindered the study of rahmnotransferases and chemoenzymatic synthesis of rhamnose-containing compounds.To address this problem,we have established an effective one-pot four-enzyme synthetic method for dTDP-Rha.The involved enzymes,including Glc-1-P thymidylyltransferase Cps23FL,dTDPGlc-4,6-dehydratase Cps23FN,dTDP-4-keto-6-deoxy-Glc-3,5-epimerase Cps23FM and dTDP-4-keto-Rha reductase Cps23FO,were derived from S.pneumonia serotype 23F and were expressed in E.coli.These enzymes were purified to be essentially homogeneous by an one step purification method using nickel column and studied in detail.The activity of Cps23FL as an enzyme to catalyze the conversion of Glc-1-P to dTDP-Glc was proved unambiguously,providing for the first time direct evidences for its biological functions.Cps23FN was proved as a dTDPGlc-4,6-dehydratase to catalyze the conversion of dTDP-Glc to dTDP-4-keto-6-deoxyglucose(dT4k6dG),also providing for the first time direct evidences for its biological functions.The dTDP-4-keto-6-deoxy-Glc-3,5-epimerase and dTDP-4-keto-L-Rha reductase activities of Cps23FM and Cps23FO were also verified by their functions to covert dT4k6dG into dTDP-Rha.Finally,based on these discoveries,an effective one-pot four-enzyme synthetic method was developed for dTDP-Rha utilizing these enztymes and commercial D-glucose-1-phosphate(Glc-1-P)as a starting material,and dTDP-Rha was obtained in an overall yield of 63%.Chapter 2 has described the enzymatic synthesis of 19 sugar nucleotides.Sugar nucleotides are the substrates used by glycosyltransferases as sugar donors to be attached to sugar acceptors.However,many sugar nucleotides are not commercialized and not easily accessible.In this work,we found that Cps23FL could accept different substrates and be used to synthesize various sugar nucleotides.Substrate specificity analysis of Cps23FL revealed that it could utilize five sugar-1-P analogous,including Glc-1-P,Gal-l-P,GlcNH-1-P,GalNH-1-P and Man-1-P,and eight natural nucleoside triphosphates,including dTTP,UTP,ATP,dATP,CTP,dCTP,GTP and dGTP,thus to produce 17 sugar-nucleoside diphosphates:dTDP-Glc,dTDP-Gal,dTDP-GlcNH,dTDP-GalNH,dTDP-Man,UDP-Glc,UDP-Gal,UDP-GlcNH,UDP-Man,ADP-Glc,dADP-Glc,dADP-GlcNH,CDP-Glc,dCDP-Glc,GDP-Glc,GDP-Man and dGDP-Glc.Plus dTDP-Rha and dTDP-4-keto-6-deoxyglucose synthesized in dTDP-Rha synthesis pathway,this study has synthesized 19 sugar nucleotides.In addition,eight Cps23FL mutants(Q24S,Q24V,Q80A,Q80E,Q80S,Q88A,Q88S and Q88T)were constructed,and it was revealed that Q88A had improved activity toward UTP up to 6 folds.Chapter 3 has provided unambiguous biochemical evidences that cps23FT encodes a β1,4 rhamnosyltransferase catalyzing the transfer of rhamnose from dTDP-rhamnose onto Glcα-PP-lipid to form Rhaβ-Glca-PP-lipid.Cps23FT was predicted to be a rhamnosyltransferase involved in the biosynthesis of S.pneumonia serotype 23 F capsular polysaccharide.However,the difficulty in accessing proper rhamnose donor and acceptor has limited studies of rhamnosyltransferases.In this study,the full length Glc-1-P transferase and its C-terminal sequence,Cps23FE and Cps23FECT,were derived from S.pneumonia serotype 23 F,expressed in E.coli,and obtained as both membrane-bound proteins and purified proteins.It was then proved that cps23FE was indeed a Glc-1-P transferase;besides,the C-terminal sequence of Cps23FE had intact Glc-1-P transferase activity.However,Cps23FE needed membrane to be active,thus the membrane-bound product,Und-PP-Glc,was not easily purifiable.Then,rhamnosyltransferase Cps23FT was derived from S.pneumonia serotype 23F,expressed in E.coli,and purified essentially to homogeneity.A chemically synthesized acceptor,Glcα-PP-(CH2)11-O-Pn,was used to characterize the activity of Cps23FT,to provide for the first time direct evidences for its rhamnose-transfering function.The disaccharide product,Rhaβ-Glcα-PP-(CH2)11-O-Pn,of this reaction was purified and obtained from dTDP-Rha in an overall yield of 40%.Substrate specificity study of Cps23FT further indicated that the pyrophosphate and the lipid moieties of the acceptor played an important role in the enzyme-acceptor recognition.Eventually,a DXD motif might be involved in the coordination with a divalent metal cation,via analysis of four Cps23FT mutants.In summary,rhamnosyltransferase and related enzymes inovolved in the CPS biosynthesis of S.pneumonia serotype 23F were studied in great detail.This research work should have a great significance.First,easy access to dTDP-Rha and other 18 other sugar nucleotides would facilitate Rha-T and other glycosyltransferase-based chemoenzymatic syntheses.Second,this research has provided for the first time direct biochemical evidences for enzymes involved in the biosynthesis pathway of dTDP-Rha and the activities of Glc-1-P transferase and rhamnosyltransferase from S.pneumonia serotype 23 F.Third,this work has established that rhamnosyltransferase may be used as a tool for chemoenzymatic synthesis of rhamnose-containing compounds,thus this research has laid a foundation for chemoenyzymatic synthesis of the repeat unit of capsular polysaccharides of S.pneumonia serotype 23F,useful in its structural study and new vaccine development. |
