title: Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Jiajun
authors.initials: J
authors.lastname: Xue
authors.affiliation: National Glycoengineering Research Center, Shandong University, Jinan 250012, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: 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.
authors.forename: Xinke
authors.initials: X
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Fengshan
authors.initials: F
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Peixue
authors.initials: P
authors.lastname: Ling
authors.affiliation: 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.
authors.forename: Juzheng
authors.initials: J
authors.lastname: Sheng
mesh_terms: D002384:Catalysis; D002809:Chondroitin Sulfates / Q000096:biosynthesis* / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002809:Chondroitin Sulfates; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli
keywords: Carbohydrate biosynthesis; Catalytic mechanism; Chondroitin polymerase; Glycosaminoglycan; Substrate specificity