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

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 similar to 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.