标题：Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor
作者：Xue, Jiajun; Jin, Lan; Zhang, Xinke; Wang, Fengshan; Ling, Peixue; Sheng, Juzheng
作者机构：[Xue, Jiajun; Zhang, Xinke; Wang, Fengshan; Sheng, Juzheng] Shandong Univ, Sch Pharmaceut Sci, Inst Biochem & Biotechnol Drug, Key Lab Chem Biol Nat P 更多
通讯作者：Ling, PX;Sheng, JZ
通讯作者地址：[Ling, PX]Shandong Acad Pharmaceut Sci, Jinan 250101, Peoples R China;[Sheng, JZ]Shandong Univ, Sch Pharmaceut Sci, Inst Biochem & Biotechnol Drug, Ji 更多
来源：BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
关键词：Carbohydrate biosynthesis; Glycosaminoglycan; Chondroitin polymerase;; Substrate specificity; Catalytic mechanism
摘要：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. (C) 2016 Elsevier B.V. All rights reserved.