Studies On One-pot Enzymatic Synthesis Of DTDP/dUDP-rhamnose

Saccharides are the most abundant organic molecules in nature,and almost all organisms can synthesize and catabolize carbohydrates.In addition to providing energy,they can also act as cellular structure and participate in a variety of life activities,such as cell recognition and migration,inflammation responses and bacterial and viral infections,etc.Saccharides,lipids,nucleic acids,and proteins are known as the four major biomolecules.Specific enzymes can catalyze the conjugation of saccharide molecules to lipids,polypeptides,and proteins to form glycoconjugates,such as glycolipids,glycopeptides,and glycoproteins.By contrast,saccharide structures are more complex.Their in vitro synthesis is an effective means to investigate the structure-function relationship of saccharides.At present,the synthesis of saccharides is still challenging,and the chemical and enzymatic methods are the two major ways in vitro.Chemical method with poor selectivity,cumbersome steps of protection and off-protection,and the difficulty in special glycosides formation??-glycosides?,is complemented effectively by biological enzymatic method.Based on the superior selectivity,enzymatic method has been applied to synthesize a wide variety of oligosaccharides,polysaccharides,and glycoconjugates.Thus far,glycosyltransferase and glycosidase are the two main members employed in saccharide synthesis in vitro.Glycosyltransferase?EC 2.4.x.y?can catalyze the transfer of activated donors,such as nucleotide sugars,to receptor molecules such as proteins,nucleic acids and lipids The product synthesized by glycosyltransferase is of superior specificity and efficiency due to the strict stereo and regio-selectivity,thus receiving much attention.However,due to the difficulties and high cost in preparation of nucleotide sugar donors,the actual application of glycosyltransferase has been limited to a certain extent.At present,the syntheses of nucleotide sugar donors are mainly performed by one-pot enzymatic methods,which avoids the tedious processes of isolation and purification,thus improving yields and reducing costs.Rhamnosyltransferase?Rha-Ts,EC 2.4.1.159?is a typical glycosyltransferase,which is widely found in a variety of bacteria and plants,and can catalyze the synthesis of rhamnose-containing compounds.The preparation of dTDP-rhamnose?dTDP-Rha?donor is challenging due to the complexity of structure,the difficulty in purification,and the containing of the special ?-Rha bond.So far,no commercial refinement is available,which has become the bottleneck of the synthesis of rhamnose-containing compounds.In the present study,the dTDP-Rha donor was synthesized successfully by the enzymatic methods.In this thesis,four DNA sequences from the genome of Saccharothrix syringae CGMCC 4.1716?NZ_JNYO01000004.1?were predicted to encode the enzymes for the dTDP-Rha synthetic pathway.They encoded glucose-1-phosphate thymidylyltransferase?SsRmlA,WP₀33428542.1,296 aa?,dTDP-D-glucose-4,6-dehydratase?SsRmlB,WP—033429095.1,351 aa?,dTDP-4-keto-6-deoxy-D-glucose-3,5-epimerase?SsRmlC,WP₀33434852.1,208 aa?and dTDP-4-keto-L-rhamnose reductase?SsRmlD,WP₀33429096.1,303 aa?,respectively.The four genes were acquired by PCR using the genome of S.syringae CGMCC 4.1716 as template,and they were ligated to pET21b or pET22b plasmids and expressed in E.coli BL21?DE3?.The heterologously expressed proteins were purified using Ni2+affinity chromatography.The activities of these four recombinases were indirectly measured by spectrophotometric method.The activity of SsRmlA was measured by analysis of OD630 through the malachite green colorimetric method.The activity of SsRmlB was measured by analysis of OD320 which reflects the production of keto groups.The activities of SsRmlC and SsRmlD were measured by analysis of OD340 which reflects the consumption of NADPH.And the active states of these four enzymes were determined using gel filtration chromatography,SsRmlA is tetramer,while SsRmlB,SsRmlC,SsRmlD were dimers.Through the quantitative analysis of enzyme activity using HPLC,SsRmlC was determined to be rate-limiting enzyme for synthesis of dTDP-Rha donor.According to the study on the substrate specificity of SsRmlA,SsRmlA can synthesize nine nucleoside-diphosphate glucoses,such as dTDP-Glc,dTDP-GlcNH2,dTDP-GlcN3,dUDP-Glc,dUDP-GlcNH2,dUDP-GlcN3,UDP-Glc,UDP-GlcNH2,and UDP-GlcN3,with Glc-1-P,Gln-1-P,and 2AzGlc-1-P as the glycosyl donor and dTTP,dUTP,and UTP as the glycosyl acceptor.Based on the investigation on the biochemical properties and the optimization of reaction conditions,SsRmlA had a Km value of 117.30 mM for Glc-1-P,and the kcat was3.46 s-1.The Km value for dTTP was 49.56 ?m,and thekcat was 5.39 s-1.Meanwhile,the enzyme activity of SsRmlA was affected largely by metal ions,which can be increased by at least 2-fold by adding Zn2+,Mn2+,Ag+,and Co2+,respectively.The influence of Mg2+was the most significant,which increased the activity of SsRmlA by 5-fold.The optimized reaction conditions of SsRmlA for dTDP-Glc synthesis were as follows:2 mM Glc-1-P,0.5 mM dTTP,1.3 mM Mg2+,pH 9.5,37? and 1.5 h,the maximum conversion of dTDP-Glc reached 93%.In the present study,two deoxynucleotide-diphosphate rhamnoses,dTDP-Rha and dUDP-Rha were synthesized by one-pot enzymatic?SsRmlA,SsRmlB,SsRmlC and SsRmlD?method using Glc-1-P as glycosyl donor,dTDP-Rha was synthized using dTTP as glycosyl acceptor,dUDP-Rha was synthized using dUTP as glycosyl acceptor.The influences of enzyme amount,substrate concentration,Mg2+concentration,NADPH concentration,temperature,pH,and reaction time on the relative content of dTDP-Rha were investigated.The optimized reaction conditions for dTDP-Rha and dUDP-Rha synthesis were as follows:2 ?M SsRmlA/B/C/D,2 mM Glc-1-P,0.5mM dTTP,1.3 mM Mg2+,1.5 mM NADPH,pH 8.5,30? and 2 h.The maximum conversion of dTDP-Rha was 65%and that of dUDP-Rha was 46%under this condition.These two products were purified using HPLC?equipped with CarboPacTM PA-100?and G10 column,and their molecular structures were identified by MS and NMR analyses.This is the first synthesis of dUDP-Rha.