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Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida to Improve the Synthesis of Heparin

  • Eduardo Stancanelli
    Eduardo Stancanelli
    Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, United States
  • Juno A. Krahn
    Juno A. Krahn
    Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, United States
  • Elizabeth Viverette
    Elizabeth Viverette
    Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, United States
  • Robert Dutcher
    Robert Dutcher
    Epigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, United States
  • Vijayakanth Pagadala
    Vijayakanth Pagadala
    Glycan Therapeutics Corporation, 617 Hutton Street, Raleigh, North Carolina 27606, United States
  • Mario J. Borgnia
    Mario J. Borgnia
    Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, United States
  • Jian Liu*
    Jian Liu
    Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, United States
    *Email: [email protected]
    More by Jian Liu
  • , and 
  • Lars C. Pedersen
    Lars C. Pedersen
    Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, United States
Cite this: ACS Catal. 2024, 14, 9, 6577–6588
Publication Date (Web):April 15, 2024
https://doi.org/10.1021/acscatal.4c00677
Copyright © 2024 American Chemical Society

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    Abstract

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    Heparin is a widely used drug to treat thrombotic disorders in hospitals. Heparosan synthase 2 from Pasteurella multocida (PmHS2) is a key enzyme used for the chemoenzymatic synthesis of heparin oligosaccharides. It has both activities: glucosaminyl transferase activity and glucuronyl transferase activity; however, the mechanism to carry out the glyco-oligomerization is unknown. Here, we report crystal structures of PmHS2 constructs with bound uridine diphosphate (UDP) and a cryo-EM structure of PmHS2 in complex with UDP and a heptasaccharide (NS 7-mer) substrate. Using a liquid chromatography–mass spectrometry analytical method, we discovered that the enzyme displays both a two-step concerted oligomerization mode and a distributive oligomerization mode depending on the nonreducing end of the starting oligosaccharide primer. Removal of seven amino acid residues from the C-terminus results in an enzymatically active monomer instead of a dimer and loses the concerted oligomerization mode of activity. In addition, the monomer construct can transfer N-acetyl glucosamine at a substrate concentration that is ∼7-fold higher than a wildtype enzyme. It was also determined that an F529A mutant can transfer an N-sulfoglucosamine (GlcNS) saccharide from a previously inactive UDP-GlcNS donor. Performing the glyco-transfer reaction at a high substrate concentration and the capability of using unnatural donors are desirable to simplifying the chemoenzymatic synthesis to prepare heparin-based therapeutics.

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