Study Of Enzymatic Synthesis Of Amino Sugar Nucleotides And Their Applications In Preparation Of Glycoconjugates

Carbohydrate (or glycans), including monosaccharide, oligosaccharide, polysaccharide and glycoconjugates are wildly existing in all living organisms. They are not only essential components of cells but also participate in various important biopathways.There are two common N-acetylhexosamines in living orgamism. They are N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). They are not only essential components of bacterial cell walls and glycosaminoglycans, but also prevalent in the core structures of glycans in glycoproteins and glycolipids. Besides, they play important roles in various metabolic processes. Such as O-GlcNAcylation involved in regulating signaling pathways, and glycolipids which have GalNAc residues involved in cellular interaction, differentiation and other processes. Studying oligosaccharides, polysaccharides and glycoconjugates which containing these two monosaccharides or their derivatives will help us to understand physiological and pathological processes as well as developing pharmaceuticals.In order to research the biological roles of glycans, we need to prepare glycans which have defined and homogeneous structures. The structures of glycans isolated from biological sources have microheterogeneity. Chemical approach for synthesizing glycans has tedious process, strict conditions and often has low yield. Enzymatic synthesis, which mimics the biosynthetic pathway, is considered more attractive with the advantages of strone regio-and stereo-specificity, high conversion percentage, and mild reaction conditions.In the enzymatic synthesis approach, Leloir type glycosyltransferases (GT) are mainly used. But there still are two unsolved problems:1) Few GT which has broad substrate specificity could be used for large scale synthesis of glycans;2) Sugar nucleotides are expensive and hard to obtain.Sugar nucleotides, also known as active sugars, are constructed by linking the anometic carbon of monosaccharides to nucleotide diphosphates or nucleotide monophosphates. They are sugar donors of Leloir type glycosyltransferases, and important precursors in biological synthesis of glycans. Analogs of natural sugar nucleotides could be used to study the structure-function relationship of glycosyltransferases and medicinal chemistry. Due to the easier and fewer steps of de novo pathway of sugar nucleotide synthesis, it has been the common stratege for preparing sugar nucleotides.One aim of this thesis is to enzymatically synthesize UDP-GalNAc/GlcNAc and their derivatives. UDP-GalNAc pyrophosphorylase from Homo sapiens (AGX1) can catalyze the generation of UDP-GalNAc from GalNAc-1-P and UTP in one step. It shows higher enzymatic activity towards GalNAc-1-P. In Chapter2, AGX1was cloned and overexpressed in Escherichia coli BL21(DE3) with a N-terminal His6-tag. After purification by Ni-column, the purity of recombinant AGX1was higher than95%. The enzyme exhibited maximum activity in pH8.5Tris-HCl buffer with the presence of10mM Mg2+at37℃. Then we systematically studied nucleotide substrate specificity of AGX1during its uridyltransfer reaction, AGX1could use dUTP and dTTP as substrates and to generate their corresponding nucleotide sugars. AGX1had a finite NTP substrates tolerance when using GalNAc-1-P and GlcNAc-1-P as its sugar-1-P substrates. From the substrate specificity of AGX1and the crystal structure, we presumed that the exocyclic oxygen04of uracil base was crucial for substrate binding and enzyme catalysis. Small changes of2’-hydroxyl group at ribose (dUTP/dTTP) make no differences in substrate recognition, but lead to slight decrease in the enzyme catalysis. When using purine nucleotide triphoshpate substrates, the enzyme-substrate binding and enzymatic activity was affected due to the large steric hindrance.Camphylobacter jejuni contains a post-translation N-glycosylation system. In Chapter3, we study the substrate specificity of a novel UDP-GlcNAc pyropyosphorylase from C. jejuni (CjGlmU), which has34%amino acid sequence similarity with UDP-GlcNAc pyrophosphoylase from E. coli K12 (EcGlmU). This enzyme show high enzymatic activity after one-step Ni-NTA purification. CjGlmU exhibited broader substrate spcificity than EcGlmU. It could use7kinds of NTPs as substrates, especially for CTP, the conversion percentage could reach62%. This enzyme prefer pyrimidine nucleotide triphosphate than purine nucleotide triphosphate. The sugar-1-P substrate specificity showed that this enzyme could tolerate N-acetyl modified GlcNAc-1-P derivatives. Finally,3kinds of unnatural sugar nucleotides were synthesized. We have set up a relatively easy strategy to prepare unnatural sugar nucleotides.Poly-N-acetyllactosamine (poly-LacNAc) is the most common and important sugar structure of cell-surface of membrane glycoconjugates which play a significant role in cell-cell interactions and cell-matrix adhesion. β-1,3-N-acetylglucosaminyltransferase (LgtA) from Helicobater pylori and Neisseria meningitidis have ever been widely used in the enzymatic and chemoenzymatic synthesis of poly-LacNAc both in vivo and in vitro. We show here the cloning and expressing of HpLgtA and NmLgtA as an N-MBP and C-His6-tagged fusion protein in E. coli. The basic characteristics of both LgtAs were systematically studied. In addition, a library of34UDP-sugars was used to study the donor substrate specificity of HpLgtA as well as NmLgtA. Overall, HpLgtA and NmLgtA have quite different tolerance toward substrate modifications. Both HpLgtA and NmLgtA can use uridine5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc), and some of its C2’-modified derivatives as donor substrates. Besides, HpLgtA could also use several derivatives with C6’-modifications. The kinetics study shows that HpLgtA has a relatively higher enzymatic efficiency than NmLgtA. The donor substrate promiscuity of HpLgtA and NmLgtA will allow efficient chemoenzymatic synthesis of diverse poly-LacNAc derivatives for binding study with galectins and for studies of their possible therapeutic applications.Hyaluronan is one kinds of important glycosaminoglycans, it takes various important parts in several biopathways. In chapter5, we use the hyaluronan synthase from Pasteurella multocida (Type A)(PmHAS) as a tool, use GlcA-Biotin as starting material for synthesizing biotinylated hyaluronan oligosaccharides by stepwise. We successfully synthesized HA disaccharide and trisaccharide. Furthmore, these HA oligosaccharides were used to study the relationship between acceptor length and PmHAS catalytic activity. PmHAS exhibited the highest activity toward trisaccharide, and showed no activit towards no modified monosaccharide:GlcA and GlcNAc. This new discovery will help us to better understand the catalytic mechanism of PmHAS.In summary, this thesis established a strategy of enzymatic synthesis and chromatographical preparation of UDP-GlcNAc/GalNAc derivatives with one enzyme from bacteria and one enzyme from human.5kinds of unnatural sugar nucleotides were prepared in vitro. We also study the sugar nuclotides substrate specificity of a novel glycosyltransferase from H. pylori. Besides, we successfully synthesized biotinylated hyaluronan oligosaccharides by stepwise stratege, also used them to study the relationship between accetpor length and PmHAS catalytic activity.