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

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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

Fingerprint

Pasteurella multocida
Transferases
Oligosaccharides
Polymerization
Uridine Diphosphate Sugars
Nitrilotriacetic Acid
Glycosyltransferases
Site-Directed Mutagenesis
Sepharose
Polymers
Research Design
heparosan

Keywords

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

Cite this

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title = "Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases",
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.",
keywords = "heparan-sulfate polymerization, molecular-weight heparin, chemoenzymatic synthesis, hyaluronic-acid, capsular polysaccharide, glycosaminoglycans, identification, synthase, glycosyltransferase, biosynthesis",
author = "A.A.E. Chavaroche and {van den Broek}, L.A.M. and C.G. Boeriu and G. Eggink",
year = "2012",
doi = "10.1007/s00253-011-3813-2",
language = "English",
volume = "95",
pages = "1199--1210",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer Verlag",
number = "5",

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Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases. / Chavaroche, A.A.E.; van den Broek, L.A.M.; Boeriu, C.G.; Eggink, G.

In: Applied Microbiology and Biotechnology, Vol. 95, No. 5, 2012, p. 1199-1210.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Synthesis of heparosan oligosaccharides by Pasteurella multocida PmHS2 single-action transferases

AU - Chavaroche, A.A.E.

AU - van den Broek, L.A.M.

AU - Boeriu, C.G.

AU - Eggink, G.

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

KW - heparan-sulfate polymerization

KW - molecular-weight heparin

KW - chemoenzymatic synthesis

KW - hyaluronic-acid

KW - capsular polysaccharide

KW - glycosaminoglycans

KW - identification

KW - synthase

KW - glycosyltransferase

KW - biosynthesis

U2 - 10.1007/s00253-011-3813-2

DO - 10.1007/s00253-011-3813-2

M3 - Article

VL - 95

SP - 1199

EP - 1210

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 5

ER -