Jiajun Xue1, Lan Jin2, Xinke Zhang1, Fengshan Wang3, Peixue Ling4, Juzheng Sheng5. 1. Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Science, Shandong University, Jinan 250012, China. 2. National Glycoengineering Research Center, Shandong University, Jinan 250012, China. 3. Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Science, Shandong University, Jinan 250012, China; National Glycoengineering Research Center, Shandong University, Jinan 250012, China. 4. Shandong Academy of Pharmaceutical Science, Key Laboratory of Biopharmaceuticals, Engineering Laboratory of Polysaccharide Drugs, National-Local Joint Engineering Laboratory of Polysaccharide Drugs, Jinan 250101, China. 5. Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drug, School of Pharmaceutical Science, Shandong University, Jinan 250012, China; National Glycoengineering Research Center, Shandong University, Jinan 250012, China. Electronic address: shengjuzheng@sdu.edu.cn.
Abstract
BACKGROUND: Currently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme. METHODS: Structurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor-acceptor pairs for KfoC. RESULTS: KfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase. CONCLUSION AND GENERAL SIGNIFICANCE: These results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.
BACKGROUND: Currently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme. METHODS: Structurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor-acceptor pairs for KfoC. RESULTS:KfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase. CONCLUSION AND GENERAL SIGNIFICANCE: These results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.