Study On The Properties And Application Of Enzymes Related To Sugar Nucleotide Synthesis Derived From Akkermansia Muciniphila

Polysaccharides and glycoconjugates are widely present in nature and participate in a variety of physiological and pathological processes.The synthesis of carbohydrate molecules is important for glycobiology research.Sugar-nucleotide diphosphates(sugar-nucleotides)are activated sugar donors in the biosynthesis of oligosaccharides and glycoconjugates.Currently,the easiest synthesis pathway to synthesize sugar nucleotides is slavage pathway which means that kinases are used to catalyze the phosphorylation of monosaccharides and corresponding pyrophosphorylase catalyze the synthesis of sugar nucleotides.Because of the limitation of kinases and pyrophosphorylase,more enzymes with broad substrate tolerance are urgently to be discovered.The thesis was divided into two parts.In the first part,the enzymatic properties of sugar-nucleotide synthesis-related enzymes derived from Akkermansia muciniphila ATCC BAA-835 were studied,and some sugar nucleotides were synthesized.In the second part,the simple synthetic route of UDP-ManNAc was explored based on the synthesis of ManNAc-1-P in the first part.Galactokinases(GalKs)could catalyze the phosphorylation of galactose(Gal)and possibly other monosaccharides.In Chapter 2,the galactokinase from Akkermansia muciniphila ATCC BAA-835(GalKAmu)was expressed and characterized.As shown in result,the enzyme showed broad substrate specificity that it could utilize 20 out of 43 monosaccharides.It had high catalytic activity towards galactose derivatives with modifications on the C-2,C-4,C-6 positions.At the same time,the galactokinase displayed activity towards Glc(52.5%),GlcNAc(15.5%),Glc-2-N3(<5%),Xyl(<5%),D-ManNAc(58%),D-ManF(37.4%)and D-ManNAz(6.5%).This was the first galactokinase isoform reported that can tolerate D-ManNAc.Additionally,the enzyme could utilize L-glucose in high yield(80%).We then synthesized several sugar-1-phosphates to identify their structures.Sugar-1-phosphates could be utilized to synthesize the sugar nucleotides under catalysis of corresponding nucleoside transferase.In Chapter 3,glucose-1-phosphate uridylyltransferase derived from Akkermansia muciniphila ATCC BAA-835(GalUAmu)was expressed and purified.Then 10 monosaccharides that could be used by galactokinase in Chapter 2 were used to detect the characterization of glucose-1-phosphate uridine transferase substrate specificity.As shown in result,the enyme could utilize 4 out of 10 monosaccharides.It could tolerant the the removal of the-OH group at the C-4 position in galactose-1-phosphate.Additionally,compared with UDP-glucose pyrophosphorylase reported,it has advantages in the synthesis of UDP-ManF.It may be because the enzyme was similar to eukaryotic-derived GalU that it has a higher degree of evolution,its relative substrate adaptability is relatively poor.We found that glucose-1-phosphate thymidylyltransferase(RmlA)derived from Akkermansia muciniphila ATCC BAA-835 had the conserved sequence G-X-G-T-R by Blast.It might be suggested that RmlA may play a role in the synthesis of sugar nucleotides.Therefore,we firstly identified the catalytic activity of RmlA in Chapter 4,and and found that RmlA has similar activity with GalUAmu in catalyzing the synthesis of UDP sugar.The result suggested that existence of RmlA might provide a basis for the structural stability of glycoconjugates of Akkermansia muciniphila ATCC BAA-835.The specific reasons have yet to be analyzed.Then,we proposed a simplified synthetic pathway of UDP-ManNAc in Chapter 4,based on the fact that galactokinase in Chapter 2 could catalyze the synthesis of ManNAc-1-P.UDP-ManNAc could be used as gly cosyl donor of capsular polysaccharide(CPS A)which was the main virulence factor of Neisseria meningitidis serotype A(NmA).It was significance for the subsequent research on sugar vaccine of NmA.UDP-sugar pyrophorylase derived from Arabidopsis thaliana,UDP-sugar pyrophorylase derived from Campylobacter jejuni and glucose-1-phosphate uridylyltransferase derived Streptococcus pneumonia were expressed and purified.Then catalytic acticity of five enzymes(including the above three enzymes,glucose-1-phosphate uridylyltransferase in Chapter 3 and glucose-1-phosphate thymidylyltransferase)for UDP-ManNAc synthesis were tested.Although the results showed that none of these five enzymes could catalyze the synthesis of UDP-ManNAc,it was believed that we can achieve the simple synthesis of UDP-ManNAc through mutations in the substrate binding center of pyrophosphorylase in subsequent studies.In summary,the thesis was focus on the properities of GalKAmu,GalUAmu and RmlA derived from Akkermansia muciniphila which related to the synthesis of sugar nucleotides.On this basis,their applications in simplifying the synthesis steps of UDP-ManNAc were explored.It laid the foundation for revealing the LPS synthesis mechanism of Akkermansia muciniphila,and was significant for the synthesis of sugar nucleotides and glycoconjugates.