Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida to Improve the Synthesis of Heparin
Year: 2024
Type: article
Abstract: (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 LC-MC analytical method, we discovered the enzyme displays both a two-step concerted oligomerization mode and a distributive oligomerization mode depending on the non-reducing end of the starting oligosaccharide primer. Removal of 7 amino acid residues from the C-terminus results in an enzymatically active monomer instead of 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 wildtype enzyme. It was also determined that an F529A mutant can transfer an N-sulfo glucosamine (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 simplify the chemoenzymatic synthesis to prepare heparin-based therapeutics.
Source: ACS Catalysis
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funders: U.S. Department of Energy, Georgia Research Alliance, National Institutes of Health, Office of Science, National Heart, Lung, and Blood Institute, National Institute of General Medical Sciences, National Institute of Environmental Health Sciences, Biological and Environmental Research, Argonne National Laboratory, Brookhaven National Laboratory, NHLBI Division of Intramural Research
Awards: G1285306620, G140363568, G1633006425, G1699008898, G171842289, G1800737955, G1867732070, Phase II SER-CAT Optimization: Acquisition of a next generation area detector, G2004402655, G2302316395, G241279073, G2632182770, G3067052642, G3262563802, G3338542329, Acquisition of a High Precision Micro Diffractometer to Support NIH funded Research at SER-CAT, G357145406, G3623357875, An Eiger2 XE 9M detector for the NYSBC-operated NYX beamline at NSLS-II, G3728080647, G434509735, G5765906131, G6153725424, G6476969910, G678799539, G6800544485, G7463374569, G7932540327, G8701900045, G9000147424, G9000152165, G9000326484, Center for Biomolecular Structure (CBMS), NIEHS Cryo-EM Core Facility, Crystallographic studies of macromolecular structures, Acquition of a microdiffractometer for SER-CAT
OpenAlex ID: W4394800995