The Application Of Arabidopsis UDP-sugar Pyrophosphorylase (AtUSP) In The Synthesis Of Sugar Nucleotide

Carbohydrates (or glycans, including monosaccharides, oligosaccharides and polysaccharides) and glycoconjugates are prevalent in all living organisms ranging from archaebacteria, bacteria to plants and animals. They are not only essential structural components, but also exhibit irreplaceable roles as informative molecules in a variety of vital biological processes. Carbohydrates store a large number of biological information due to their complex and volatile structures. But the microheterogeneity of the carbohydrate chains separated from natural glycan increase the difficulty on sugar biological research. The gain of glycan with uniform structure has become the precondition for the research on their structure and function.The chemical pathway to synthesize glycoconjugates is tedious and often has low yields. In contrast, glycosyltransferases in the biological synthetic pathway of glycans can efficiently form regio-specific and stereo-specific glycosidic bonds. Therefore, synthesis of structure-defined glycans by glycosyltransferases (mainly Leloir-type glycosyltransferases) has become an attractive alternative to the chemical synthesis. Nucleotide sugars are the sugar donors of glycosyltransferases in synthesis of glycoconjugates, but the exorbitant price of nucleotide sugars restricts the biological synthetic pathway.This thesis is divided into two main parts:the first part is the synthesis of nucleotide sugars by one-pot pathway; the second is the study on nucleotide triphosphate secificity of AtUSP, and the calculation of enzymatic reaction constant on AtUSP towards UTP,dUTP and dTTP. Besides we purified dUDP-Glc and dTDP-Glc in small scale.In the in vitro synthesis of sugar nucleotides, the simplest strategy is using the salvage pathway. In salvage pathway, the monosaccharides will be firstly phosphorylated to the form of sugar-1-phosphate, which will subsequently be catalyzed to sugar nucleotides by pyrophosphorylase or uridylyltransferase. In chapter2, the AtUSP gene was cloned from the total cDNA of Arabidopsis thaliana. The active AtUSP was recombined and expressed in E.coli BL21(DE3). Combined with a galactosekinase from Streptococcus pneumoniae (SpGalK) which had been reported in our research group and a commercial available pyrophosphatase from yeast (PPase), we succeed in synthesizing nucleotide sugars by one-pot pathway.Both SpGalK and AtUSP have broad substrate tolerance, they could catalyze chemically modified monosaccharides. Eight natural or chemically modified monosaccharides were tested in the one-pot reaction system. Among the five nucleotide sugars synthesized by the one-pot reactions UDP-Gal and UDP-4-N3-Gal got high conversion rate. It was95%for UDP-Gal and43%for UDP-4-N3-Gal, respectively. Both UDP-Glc and UDP-Fuc got lower conversion rate under30%, they were23%and29%respectively. The conversion rate for UDP-2-N3-Gal was the lowest in the five nucleotide sugars. It can only be detected in MS. In chemical reaction, the-N3is an active group. We synthesized UDP-4-N3-Gal with high conversion rate in one-pot reaction. This lay a material foundation for the synthesis of C-4modified UDP-Gal by chemical method. Compared with the UTP-Glc uridylyltransferase from S. pneumoniae (SpGalU), AtUSP has more tolerance towards C-4modified Gal-1-P. This enzyme is suited to synthesize C-4modified UDP-Gal.In chapter3, the nucleotide triphosphate specificity of AtUSP was systematically studied. Among the nine nucleotide triphosphates tested in our assays, five of them were pyridine-nucleotide triphosphates (UTP, dUTP, dTTP, CTP and dCTP), four of them were purine-nucleotide triphosphates (ATP, dm6ATP, GTP�'�dPdGTP). Three nucleotide triphosphates could be catalyzed by AtUSP, namely UTP, dUTP and dTTP. The percent conversion of UTP was the greatest in the three nucleotide triphosphates. It reached97%. The percent conversion of dUTP and dTTP were85%and84%, respectively. The other six nucleotide triphosphates could not be catalyzed by AtUSP. This maybe related to the hydrogen bond formed between nucleotide triphosphates and AtUSP. The O-4on the pyridine ring of UTP, dUTP and dTTP could form hydrogen bonds with AtUSP. This promotes the incorporation between nucleotide triphosphates and AtUSP. When CTP or dCTP was used as nucleotide substrate, oxygen O-4was replaced by an amino group. This modification affected the enzyme-substrate binding and caused the loss of enzyme catalysis. For the purine-nucleotide triphosphates, the large structure base increased the steric hindrance of themselves. And this becomes another limiting factor for the enzyme catalysis. However, the hydrogen bond constructed between2’-hydroxyl group on the ribose group and AtUSP can not affect the recognition to the nucleotide triphosphates. It just leads to slight decrease in the enzyme catalysis. The enzymatic constants showed that the values for Vmax、kcat and&kcat/Km of AtUSP towards UTP outclass that for dUTP and dTTP. This indicates that the optimum nucleotide triphosphate substrate was UTP. After that, dUDP-Glc and dTDP-Glc were prepared in small scale. With the applying of alkaline phosphatase, the remanent nucleotide triphosphates in the reaction were degraded. This simplifies the purification procedure of nucleotide sugars. High purity dUDP-Glc and dTDP-Glc were separated by a one step gel exclusion chromatography.