Systematic Study On GlcNAc-1-P Uridyltransferase From Escherichia Coli K12

Glycans in the form of oligosaccharides and glycoconjugates(glycolipids,glycoproteins,etc ) are vital biopolymers found in organisms across all domains of life.They play critical roles in mediation of numerous complex biological processes.Specific changes in glycan profiles have been associated with certain disease states such as cancer and inflammation,illustrating the potential of using glycans in clinical diagnosis and perhaps as targets to develop therapeutics.The biosynthesis of saccharides in nature uses glycosyltransferase.They transfer a given monosaccharide from the corresponding sugar nucleotide(sugar donor) to a specific hydroxyl group of a sugar acceptor.With obvious advantages of achieving high regio- and stereochemistry of glycosidic bonds,often in a high efficient manner,glycosyltransferase-catalyzed enzymatic synthesis of saccharides becomes an attractive and powerful alternative to the chemical synthesis. Oranic synthesis could give targets in therapeutics development by synthesizing complex analogs using nature compounds.Chem-enzyme synthesis becomes an important research way and mean in the research field of Glycobiology and Glcochemistry by combining bio-enzyme and organic methods exploiting their own parricular advantages for mutual benefit.Nucleotide sugars,also call active sugars,are compounds which are synthesized using reducing end of monosaccharides and the ending phosphate group of nucleotide monophosphate or bisphosphate.The biological importance for those compounds:1 to product necessary active saccharide for glycosyltransferase reaction by transformation among nucleotide sugars;2 to act as glycosyl donors in glycan and polysaccharides biosynthesis pathway.Uridine 5'-diphosphate N-acetylglucosamine(UDP-GlcNAc),the key cytoplasmic amino nucleotide sugar,is an essential precursor of the biosynthesis of various cell components including cell wall peptidoglycan,lipopolysaccharides,enterobacterial common antigens,chitin, glycosylphophatidylinositol anchors,glycosaminoglycans and glycoproteins.UDP-GlcNAc was synthesized by a multi-enzymatic reaciton using fructose-6-P which produced during glycolysis acts as starting substrates in in vivo UDP-GlcNAc synthesis pathway. Two variations of this pathway exist,one in eukaryotes and one in prokaryotes.The main difference between two pathways is the acetylation and isomerization order for glucosamine-6-P. In the eukaryotic pathway,fructose-6-phosphate produced during glycolysis is transaminated and isomerized to glucosamine-6-phosphate,followed by acetylation to GlcNAc-6-phosphate, isomerization to GlcNAc-1-phosphate.In prokaryotes,glucosamine-6-phosphate is isomerized to glucosamine-1-phosphate,followed by its N-acetylation to N-acetyl-D-glucosamine-1-phosphate.The aim of this thesis is exploring the bifunctional GlcNAc-1-P uridyltransferase (GlcNAc-1-P pyrophosphorylase,GlmU) and its uridyltransferase domain(GlmU-Tr229) from Escherichi coli K12 for systematic study on broad substrate specificity.In addition to the biochemical study towards the illumination of their substrate adaptability,enzymatic dynamics and catalytic mechanism,the synthetic application of this enzyme was also elucidated by the synthesis of two amino nucleotide sugars in small scale.Pilot study on the GlmU site-directed muation were also carried out and got some progress.In chapter 2,GlmU was cloned from the genome of E.coli K12 and was over-expressed in E. coli BL21(DE3).The recombinant His-tagged GlmU protein was purified by Ni-NTA column. SDS-PAGE showed that the purity of GlmU protein was more than 90%,the apparent molecular weight of the GlmU protein was about 50 kDa,consistent with the calculated mass.The effect of N-acetyl group GlcNAc on GlmU enzymatic reaction was well investigated using Glc-1-P(C2 group OH),GlcNAc-1-P(C2 group N-acetyl) and GlcNAcZ-1-P(C2 group N-adize-acetyl group).GlmU exhibited activity for all three sugar-1-Ps with diverse yields.The yields for GlcNAcZ-1-P decreased to 86%of that for GlcNAc-1-P with UTP as pyrophosphate donor.While the conversion efficiencies for Glc-1-P which lacks the C2 N-acetyl group dropped to around 30%of this for GlcNAc-1-P.Previous study suggested that GlcNAc-1-P interacted with the enzyme by hydrogen bonds between the N-acetyl arm and Thr82 and Glu154.The significant decline of GlmU activity for Glc-1-P compared with that for GlcNAc-1-P was consistent with the structure study,which demonstrated the important role of Thr82 and Glu154 residues in sugar-1-P recognition.In contrast,GlmU-Tr229 had better tolerance for the modifications on the N-acyl group,as indicated by the result for GlcNAcZ-1-P.In chapter 3,bifunctional GlmU N-terminal uridyltransferase domain(GlmU-Tr229) was cloned and expressed in E.coli BL21(DE3) according to the crystal structure of GlmU with a 55 mg per Liter souble protein expression level.To investigate the substrate specificity towards NTP, GlcNAc-1-P was used as the sugar-1-P substrate.Twelve NTPs were included into the reaction system,respectively.Capillary electrophoresis(CE) was used to detect the formation of NDP-GlcNAcs.The yields of NDP-GlcNAcs indicated a substrate tolerance of GlmU-Tr229 for NTPs in the following order:UTP＞dUTP＞dTTP＞＞CTP＞dATP/dm⁶ATP,suggesting that GlmU-Tr229 prefers pyrimidine nucleotide than purine nucleotide.The crystal structure of GlmU showed that the active site pocket of the pyrophosphorylase domain was bound by two lobes.The first lobe(residues Asn3-Val111 and His216-227) included strandsβ1-β4,which interacted primarily with the nucleotide moiety of UDP-GlcNAc.The second lobe(residues Glu112-Val215) included strandsβ5-β7,which interacted primarily with GlcNAc portion of the sugar nucleotide.Uracil base was recognized by a hydrogen bond between its ring N3 and Gln76 residue,and by two hydrogen bonds between its exocyclic oxygen O4 and Gly81 and Gln76,respectively.The ribose group was recognized by a hydrogen bond between its 2'-hydroxyl group and Gly14.Our results indicated that pyrophosphorylase domain of GlmU had a notable substrate tolerance,small changes on ribose 2'-hydroxyl group(dUTP) or uracil base C5 (dTTP) do not affect the pyrophosphorylase activity.The hydrogen bond between ribose group 2'-hydroxyl group and Gly14 was not critical for substrate recognition.The first lobe which contained the pyrophosphorylase active site was suitable to bear the existence of a methyl group in uracil base C5.To demonstrate the application of the recombinant GlmU-Tr229 for the synthesis of UDP-GlcNAc analogs,we performed a synthetic reaction in multiple mg scale for dUDP-GlcNAc and UDP-GlcNAcZ.Mono Q ion-exchange column(GE Healthcare) was used to isolate the products from the reaction mixture.The products were further desalted by P2 gel filtration column (Bio-rad).The isolated dUDP-GlcNAc(5.1 mg,57.6%) and UDP-GlcNAcZ(4.3 mg,44.4%) were identified by ESI-MS and NMR spectroscopy.We also investigated biochemical characters of GlmU-Tr229.Our results demonstrated that divalent metal was necessary for GlcNAc-1-P uridyltransfer reaction,which could be used as GlmU cofactor.The dependent relationship of divalent metal ranked:Co²⁺＞Mn²⁺＞Mg²⁺＞＞Zn²⁺ /Cu²⁺/Ni²⁺＞EDTA.The optimal divalent metal cocentration for the reaction was 5 mM when Mg²⁺ was used as cofactor.GlmU-Tr229 catalyzed GlcNAc-1-P uridyltransfer reaction under pH6.5-8.5 with an optimal pH 7.5.In addition,the product inhibition effect of the reaction byproduct inorganic pyrophosphate(IPP) was studied.Pyrophosphatase which catalyzes the hydrolysis of IPP to two orthophosphates was omitted to the reaction mixture.UDP-GlcNAc conversion ratio decreased to 65%in contrast with 95%at maximum when pyrophosphatase existed in the reaction.The result showed inhibition of IPP to the reaction and hydrolysis of IPP by pyrophosphatase could facilitate the uridyl transfer reaction.In chaper 4,GlcNAc-1-P was synthesized by a N-acetylhexosamine 1-kinse from Bifidobacterium longum using ATP and GlcNAc as substrates in in vitro reaction.The product GlcNAc-1-P was then separated by organic method using a sialic gel column.Lack of GlcNAc-1-P could be settled using this synthsis pathway in GlmU enzymatic study.To further study on nucleotide triphosphate substrate specificity of the E.coli K12 GlmU-Tr229,site-directed mutaions were done in residues Gln76 and Gly81 of GlmU-Tr229. Biochemical characters of one of the mutations GlmU-Tr229 Q76E was well studied using UTP and CTP as nucleotide triphosphate,respectively.Capillary electrophoresis profiles showed that GlmU-Tr229 Q76E had a notable change in nucleotide triphosphate substrate specificity.CTP could be recognized and used in the formation of CDP-GlcNAc with a yield above 50%.However, the conversion ratio for UTP reactions was decreased to 50%in contrast with 95%at mixmum.in GlmU reaction.This work is still ongoing.In sumary,this work provided a feasible approach for the synthesis of UDP-GlcNAc analogs which can be used to study GlcNAc-transferase reaction and various biosynthesis pathways for cell components.The systematic study on the broad substrate specificity of GlmU and GlmU-Tr229 from E.coli K12 could help us to enhance the basic knowledge of enzyme realted to amio-sugar nulceotide synthesis.