Impact Of Donor Binding On Polymerization Catalyzed By KfoC By Regulating The Affinity Of Enzyme For Acceptor

Chondroitin sulfate proteoglycans (CSPGs) are key members of the proteoglycan family, which play functional roles in biological processes. They have been implicated in many diseases, such as Alzheimer, cancer, osteoarthritis and so on. Chondroitin sulfate (CS) links to the core protein as a side chain of CSPGs. CS backbone consists of a disaccharide repeating unit including D-glucuronic acid (GlcUA) and N-acetyl-galactosamine (GalNAc) with β 1-3 and β 1-4 linkages.CS have been used as important drugs in many fields. A number of marketed CS are isolated from animal sources, such as shark cartilage or bovine. Therefore, most of commercial CS are structurally quite heterogeneous in size and sulfation, causing potential safety problems and limiting further development. It is highly desirable to study effective synthetic strategies to produce homogeneous CS. Chemoenzymatic synthesis could be an effective method to combat such problems and it had leapt forward in these years. A series of structurally defined heparin (HP) oligosaccharides had been synthesized by chemoenzymatic methods with efficiency. Thus, full understanding of the substrates and catalytic mechanisms of enzymes are essential in structurally defined oligosaccharide synthesis.Fortunately, some bacteria, including Escherichia coli strain K4, Pasteurella multocida type F and Avibacterium paragallinarum genotype I possess CS backbone, which are similar to human and animal CS or its derivatives. However, the amount of polysaccharide produced by these strains is not suitable to be used as a commercial platform. Nowadays, a series of microbial-derived chondroitin polymerases have been investigated which making it possible to produce homogeneous CS oligosaccharides. So it is necessary to investigate the mechanism for microbial biosynthesis of CS and the substrate specificities of CS backbone polymerase.E.coli K4 capsular polysaccharide consists of a GlcUA and GalNAc repeating disaccharide unit to which a fructose residue linked at C-3 position of GlcUA residues. Previous study had shown that the CS backbone polymerase is KfoC. KfoC has two glycosyltransferase activities, including N-acetylgalactosaminyltransferase (GalNAc-T) and glucuronyltransferase (GlcUA-T). KfoC could transfer UDP-GalNAc or UDP-GlcUA to the non-reducing end of oligo-or poly-acceptors. However, the understanding of the substrate tolerance of KfoC is incomplete. In this study, some structurally defined oligosaccharides, including HP backbone oligosaccharide and its N-modified derivatives, HA tetrasaccharide and CS backbone were prepared to be used as the acceptor substrates. Interestingly, we found that all of these GAG oligosaccharides could be served as the acceptors of KfoC. Some UDP-sugars and their analogs were used to investigate donor substrate specificities and it is finally confirmed that KfoC had broader donor specificities. At last, enzyme-substrate interactions were studied, especially the effects of initial substrate binding on the affinity of secondary substrate for enzyme-substrate complex. A variety of new interesting findings were born in these experiments. Our results show that binding of donor molecules regulate the affinity of KfoC for acceptor molecules and the length of acceptors would somewhat influence the polymerase reaction and elevated monosaccharide transfer efficiency could occur as the saccharide chain became longer. Besides, the findings of subsequent injections may support the hypothesis of DeAngelis that UDP-sugars bind to the glycotransferase first during the polymerization. These results could help in the development of chemoenzymatic synthesis approaches towards chimeric GAG oligosaccharides. They could also be helpful in designing further strategies for directed evolution of KfoC to obtain more mutants with broader substrate specificities and higher catalytic activities.