Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases

A.A.E. Chavaroche, L.A.M. van den Broek, C.G. Boeriu, G. Eggink

Research output: Contribution to journalArticleAcademicpeer-review

16 Citations (Scopus)

Abstract

Pasteurella multocida heparosan synthase PmHS2 is a dual action glycosyltransferase that catalyzes the polymerization of heparosan polymers in a non-processive manner. The two PmHS2 single-action transferases, obtained previously by site-directed mutagenesis, have been immobilized on Ni(II)-nitrilotriacetic acid agarose during the purification step. A detailed study of the polymerization process in the presence of non-equal amounts of PmHS2 single-action transferases revealed that the glucuronyl transferase (PmHS2-GlcUA+) is the limiting catalyst in the polymerization process. Using experimental design, it was determined that the N-acetylglucosaminyl transferase (PmHS2-GlcNAc+) plays an important role in the control of heparosan chain elongation depending on the number of heparosan chains and the UDP-sugar concentrations present in the reaction mixture. Furthermore, for the first time, the synthesis of heparosan oligosaccharides alternately using PmHS2-GlcUA+ and PmHS2-GlcNAc+ is reported. It was shown that the synthesis of heparosan oligosaccharides by PmHS2 single-action transferases do not require the presence of template molecules in the reaction mixture.
Original languageEnglish
Pages (from-to)1199-1210
JournalApplied Microbiology and Biotechnology
Volume95
Issue number5
DOIs
Publication statusPublished - 2012

Keywords

  • heparan-sulfate polymerization
  • molecular-weight heparin
  • chemoenzymatic synthesis
  • hyaluronic-acid
  • capsular polysaccharide
  • glycosaminoglycans
  • identification
  • synthase
  • glycosyltransferase
  • biosynthesis

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