Mechanism Of Substrate Selectivity For Escherichia Coli K4 Uridine Diphosphate Glucose-4-Epimerase(KfoA)

Glycosaminoglycans(GAGs)which are an unbranched polysaccharide with a high negative charge often present in the cytoplasm in the free state or covalently bound to proteins and anchored to the surface of cells in the form of glycoproteins.With the help of multiple protein receptor and chemokines,GAGs play a crucial role in many physiological processes such as cell adhesion,inflammatory response,and development.Due to its high viscosity and low compressibility,chondroitin sulfate(CS),an important branch of GAGs,becomes the ideal synovial fluid joint cavity and eye and is commonly used in the treatment of arthritis and dry eye.However,with the deepening of research,more and more functions of CS are being explored.Currently,commercially available CS is mostly obtained from separation and extraction of animals’tissue.There are many disadvantages in this process,such as non-uniform products,pollution of impurity.All of these restrict the application of CS.Therefore,it is very urgent to efficiently produce a structurally uniform CS through developing a new method of non-animal-sourced production.E.coli 05:K4:H4(ATCC 23502),also known as K4,is an uropathogenic strain,and its capsular polysaccharide is often called K4CPS which is belonged E.coli group 2 capsules.It is formed from a chondroitin-repeat disaccharide unit consisting of GlcA β(1-3)-GalNAc β(1-4)to which fructose is β-linked at position C-3 of the GlcA residue.So it is a potential chondroitin fermentation producing strain.However,the production of wild-type strains of polysaccharides cannot meet commercial demand.Therefore,we studied the genes involved in the synthesis of capsular polysaccharides in this strain,which is of great significance for the microbial fermentation and chemical enzymatic synthesis of CS in the future.The whole genome of E.coli 05:K4:H4 has been sequenced.But the biochemical characteristics of KfoA encoded by the K4 CPS gene cluster have not been explicitly studied in vitro.However,through the amino acid sequence alignment of KfoA(Accession No.CCQ00728.1)and GalE(Accession No.CCQ02734.1)which is a UDP-Glucose-4-epimerase of E.coli,it was found that KfoA and GalE-E.coli have approximately 60%similarity,which is high homology.Thus,we hypothesize that KfoA is responsible for the supply of UDP-GalNAc through the transform of UDP-GlcNAc in the K4CPS synthesis.Therefore,we have conducted this in-depth study of the substrate characteristics and mechanism of KfoA.Firstly,by constructing the pET28a(+)-His-KfoA recombination expression system in vitro,we realized soluble high-level expression of KfoA and established stable steps of separation and purification to obtain a large amount of KfoA.At the same time,its UDP-GlcNAc/Glc-4-epimerases activity was determined by off-line detection of capillary electrophoresis.Secondly,due to the little differences among the structure of these four uridine diphosphate monosaccharides,which caused difficulties in detection,we established two new rapid detection methods,one is capillary electrophoresis on-line detection of UDP-GlcNAc/Glc-4-epi Activity which is based on the EMMA method;another is coupling chondroitin polymerase(KfoC),acetylated substrate 4 epimerase activity assay.The establishment of these new activity detection methods makes detection more convenient,while achieving high efficiency and low cost of activity detection.In addation,the in vitro reactions catalyzed by the coupling of KfoA(or GalE-E.coli)and KfoC mimic the in vivo elongation of K4CPS to a certain extent.Thus,our results provide direct evidence that KfoA is a higher efficiency UDP-GalNAc provider than GalE-E.coli during K4CPS biosynthesis in vivo.Finally,by enzymatic kinetics and conversion experiments,compared to UDP-Glucose,recombinant KfoA exhibited a clear preference for acetylated substrate(UDP-GlcNAc)in vitro.Moreover,when using UDP-GlcNAc as a substrate,KfoA is much greater than GalE-E.coli in the catalytic efficiency,affinity,and maximum reaction rate.However,after alignment of the amino acid sequences of KfoA and UDP-Glc-4-epi family members,it was found that,compared to UDP-Glc-4-epimerases which is prevalent in each subtype E.coli,amino acid sequence between KfoA and group 2 from GalE family members are the highest similarity and best homology.However,KfoA has a stronger preference for acetylated substrates(UDP-GlcNAc)than any other group 2 members,and this substrate selectivity is similarity to group 3 members.So in order to explore the molecular mechanism of this unique substrate preference of KfoA,we analyzed the amino acid sequence of KfoA and other members of the GalE family again.Through the analysis of the amino acid sequence alignment with other members of the GalE family and the analysis of the selectivity of the mutant KfoA identified the key amino acids why acetylate substrate preference is determined.Through the use of homology modeling and molecular docking,we preliminary revealed the molecular mechanisms that affect the selectivity of KfoA by related amino acid.In summary,we recombinantly expressed UDP-Glc-4-epimerase(KfoA)which is encoded by a gene from region 2 of the K4 capsular gene cluster and determined its UDP-GlcNAc/Glc 4 epimerase(UDP-GlcNAc/Glc-4-epi)activity by CE off-line detection in this study.At the same time,two new types of rapid detection methods for UDP-GlcNAc/Glc-4-epi activity were established to achieve high efficiency and low cost.Through enzymatic kinetics and conversion experiments,combined with bioinformatics methods such as protein bioinformatics and molecular docking,it was determined that KfoA has a strong preference for acetylated substrates,and initially revealed how the relevant amino acid affects the selectivity and molecular mechanism of substrate of KfoA,providing a theoretical basis for the subsequent transformation of the GalE family(including KfoA)as the tool enzyme in production.