| Xylose is a pentose,a pentaaldehyde,a component of xylan,which is widely found in plant cells and also can be found in animal heparin,chondroitin and glycoproteins.At present,free xylose has not been found in nature,and xylose-related glycoconjugates have been found having important biological values.Such as,they can be the extracellular polysaccharide components of cell wall in plants and bacteria,provide the function of intercellular communication,be involved in signal transduction and communication between cells,cell proliferation,tumor cell growth.During the synthesis of xylose conjugates,a large number of xylose nucleotides are required as sugar donors.UDP-xylose is one of the nine kind of nucleotide sugars that commonly found in living organisms,plays an irreplaceable role in the nucleotide sugar synthesis of the organism.The synthesis pathway of UDP-xylose is mainly as follows:xylose can produce the substrates(Xyl-1-P)that required for UDP-xylose synthesis under the catalization of related kinases,Xyl-1-P can react with UTP under the catalysis of Xyl-1-phosphate thrimidylytransferase to produce UDP-Xyl.Normally,chemical method is used to produce UDP-Xyl in industry,but this method has some disadvantages,such as complicated steps,low yield and high cost.The bio-enzyme engineering method for synthesizing of UDP-Xyl has the advantages of easy operation,pure products,low cost,high yield,and environmental friendliness,but it is required high-performance enzymes.At present,there are few studies on xylose-1-phosphate thymidylytransferase in the above pathways.In this paper,we use the required sugar-1-phosphate nucleoside transferase enzymes in the synthesis pathway of Xyl-1-P to UDP-Xyl as the research object,excavate xylose-1-phosphate thymidylytransferase enzyme with high performance and study its enzymatic characteristic and protein structure.In this thesis,a gene may encode glucose-1-phosphate thymidylytransferase was discovered from Terriglobus roseus.After gene cloning,vector construction and induction in vitro,we obtain an active enzyme.We found it has the activity of xylose-1-phosphate thymidylytransferase.The optimal pH,temperature and other reaction conditions were studied.At the same time,in order to explain the catalytic mechanism,the condition of protein crystalization was studied and the three-dimensional structure was analyzed.The specific research content is as follows:1.Gene cloning,recombinant expression,purification and enzymatic activity detection of novel xylose-1-phosphate thymidylytransferase Tr0678In this study,through bioinformatics sequence alignment,a gene may encoding glucose-1-phosphothymidylytransferase(Tr0678)was discovered in Terriglobus roseus(DSM 18391)which is a kind of genus Acidobacteria.By designing primers,PCR amplification,vectors construction,the gene of recombinant the sugar-1-thymidine phosphotransferase was obtainned.The plasmid was transfered into E.coli BL21(DE3)cells for expression.The histidine-tagged recombinant protein was seperated and purified with a nickel affinity chromatography,a single band of the target protein with higher purity was identitied by SDS-PAGE analysis,and the band size was basically the same as the theoretical molecular weight of 33 kDa.An improved Bradford protein concentration assay kit was used to determine its protein concentration.The actual concentration of purified protein was 3.3573±0.25 mg/mL.To determine the activity of this sugar-1-phosphate thymidylytransferase,we use Xyl-1-P as substrate,UTP was added to the reaction system,and the reaction was performed at 37?.The supernatant after the reaction was analysized via liquid mass spectrometry to detect the reaction products.According to the mass spectrum peaks,it was preliminarily judged that Tr0678 has relevant activity.Based on the molecular weight,the product was determined to be UDP-Xyl,it is sure that Tr0678 has significant activity on Xyl-1-P,the enzyme is determined to be xylose-1-phosphate thymidylytransferase.Next,in this study,Xyl-1-P was used as the substrate,and the established LC-MS detection method was used to explore the biochemical characterization of the xylose-1 phosphate thymidylytransferase.The results showed that the optimal reaction pH of the enzyme was 8.0.The optimal reaction temperature is 50?,but it can maintain high activity between 37? and 50?.2.Study on the activity sites,crystallization screening and sturacture analysis of the novel xylose-l-phosphate thymidylytransferaseSome gene sequences of sugar-1-phosphate thymidylytransferase with similar functions was selected.By multiple sequence alignment,it was found that the aspartic acid at position 108 is the key amino acid that affects the activity of Tr0678 enzyme.A site-directed mutation was did to mutate it to alanine and asparagine.By comparing the relative enzyme activity of the mutants with wild-type protein,it was found that the relative enzymatic activity had almost lost after the mutation at position 108.Therefore,Asp 108 is thought to be the key amino acid that affects the catalytic activity of the xylose-1-phosphate thymidylytransferase.Through the large-scale expression and purification of Tr0678 protein,a protein solution with higher purity was obtained,and preliminary screening of crystallization conditions was performed.In a condition of 0.1 M citric acid buffer pH 5.0,20%(v/v)PEG6000,mutant protein crystal was found.Based on wild-type protein preliminary screening conditions,the protein buffer was changed to citric acid-sodium sulfate buffer with the pH is 6.5,then the crystal is optimized by reducing the concentration to obtain optimized wild-type protein crystals.By adding 1%substrate,the wild-type and mutant Tr0678 were co-crystallized with the substrate to obtain a complex crystal.The protein crystal has a resolution of 2.65 (?) after X-ray diffraction.Then the diffraction data was collected and analyzed,and the protein structure of Tr0678 was analyzed at last.The structure of the mutant and substrate complex was analyzed using this structure as a template.An active unit of the protein was found to be a dimer of the protein,and the two proteins were associated with each other to get the activities.Further studies are needed on the catalytic mechanism of this enzyme. |
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