Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

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

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Abstract

Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars
Original languageEnglish
Pages (from-to)1777-1785
JournalJournal of Biological Chemistry
Volume286
Issue number3
DOIs
Publication statusPublished - 2011

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Uridine Diphosphate Sugars
Pasteurella multocida
Glycosyltransferases
Hydrolases
Polymerization
Uridine Diphosphate
Enzymes
Transferases
heparosan synthase
heparosan
Mutagenesis
Molecules
Monosaccharides
Site-Directed Mutagenesis
Chain length
Hydrolysis
Polymers

Keywords

  • hyaluronan synthase
  • chemoenzymatic synthesis
  • identification
  • biosynthesis
  • glycosidases
  • transferase
  • mechanism
  • polymers
  • distinct
  • sulfate

Cite this

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title = "Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity",
abstract = "Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUA{\ss}1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars",
keywords = "hyaluronan synthase, chemoenzymatic synthesis, identification, biosynthesis, glycosidases, transferase, mechanism, polymers, distinct, sulfate",
author = "A.A.E. Chavaroche and {van den Broek}, L.A.M. and J. Springer and C. Boeriu and G. Eggink",
year = "2011",
doi = "10.1074/jbc.M110.136754",
language = "English",
volume = "286",
pages = "1777--1785",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology",
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TY - JOUR

T1 - Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity

AU - Chavaroche, A.A.E.

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

AU - Springer, J.

AU - Boeriu, C.

AU - Eggink, G.

PY - 2011

Y1 - 2011

N2 - Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars

AB - Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars

KW - hyaluronan synthase

KW - chemoenzymatic synthesis

KW - identification

KW - biosynthesis

KW - glycosidases

KW - transferase

KW - mechanism

KW - polymers

KW - distinct

KW - sulfate

U2 - 10.1074/jbc.M110.136754

DO - 10.1074/jbc.M110.136754

M3 - Article

VL - 286

SP - 1777

EP - 1785

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 3

ER -