8VIW | pdb_00008viw

Cryo-EM structure of heparosan synthase 2 from Pasteurella multocida with polysaccharide in the GlcNAc-T active site

PDB DOI: https://doi.org/10.2210/pdb8VIW/pdb
EM Map EMD-43269: EMDB EMDataResource

Classification: TRANSFERASE
Organism(s): Pasteurella multocida
Expression System: Escherichia coli
Mutation(s): No

Deposited: 2024-01-05 Released: 2024-07-24
Deposition Author(s): Krahn, J.M., Pedersen, L.C., Liu, J., Stancanelli, E., Borgnia, M., Vivarette, E.
Funding Organization(s): National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)

Experimental Data Snapshot

Method: ELECTRON MICROSCOPY
Resolution: 3.30 Å
Aggregation State: 2D ARRAY
Reconstruction Method: SINGLE PARTICLE

wwPDB Validation 3D Report Full Report

This is version 1.1 of the entry. See complete history.

Literature

Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida (PmHS2) to Improve the Synthesis of Heparin.

Stancanelli, E., Krahn, J.A., Viverette, E., Dutcher, R., Pagadala, V., Borgnia, M.J., Liu, J., Pedersen, L.C.

(2024) ACS Catal 14: 6577-6588

PubMed: 39990868 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1021/acscatal.4c00677
Primary Citation of Related Structures:
8VH7, 8VH8, 8VIW

PubMed Abstract:
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 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.

Organizational Affiliation

Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA.

Macromolecule Content

Total Structure Weight: 265.98 kDa
Atom Count: 18,180
Modelled Residue Count: 2,180
Deposited Residue Count: 2,236
Unique protein chains: 1

Macromolecules

Find similar proteins by:

(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Heparosan synthase B	A, B, C, D	559	Pasteurella multocida	Mutation(s): 0 Gene Names: hssB
UniProt
Find proteins for Q5SGE1 (Pasteurella multocida) Explore Q5SGE1 Go to UniProtKB: Q5SGE1
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	Q5SGE1
Sequence Annotations Expand
Reference Sequence LOADING

Oligosaccharides

Help

Entity ID: 2
Molecule	Chains	Length	2D Diagram	Glycosylation	3D Interactions
beta-D-glucopyranuronic acid-(1-4)-2-deoxy-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid-(1-4)-2-deoxy-2-(sulfoamino)-alpha-D-glucopyranose-(1-4)-beta-D-glucopyranuronic acid	E, F, G, H	5		N/A	Interactions Focus chain E Focus chain F Focus chain G Focus chain H Interactions & Density Focus chain E Focus chain F Focus chain G Focus chain H
Glycosylation Resources
GlyTouCan: G86319LQ GlyCosmos: G86319LQ

Small Molecules

Ligands 2 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
UDP (Subject of Investigation/LOI) Query on UDP Download Ideal Coordinates CCD File SDF format, chain L [auth A] SDF format, chain R [auth C] SDF format, chain T [auth C] SDF format, chain J [auth A] SDF format, chain N [auth B] SDF format, chain P [auth B] SDF format, chain V [auth D] SDF format, chain X [auth D] MOL2 format, chain L [auth A] MOL2 format, chain R [auth C] MOL2 format, chain T [auth C] MOL2 format, chain J [auth A] MOL2 format, chain N [auth B] MOL2 format, chain P [auth B] MOL2 format, chain V [auth D] MOL2 format, chain X [auth D] mmCIF format, chain L [auth A] mmCIF format, chain R [auth C] mmCIF format, chain T [auth C] mmCIF format, chain J [auth A] mmCIF format, chain N [auth B] mmCIF format, chain P [auth B] mmCIF format, chain V [auth D] mmCIF format, chain X [auth D]	J [auth A] L [auth A] N [auth B] P [auth B] R [auth C] J [auth A], L [auth A], N [auth B], P [auth B], R [auth C], T [auth C], V [auth D], X [auth D]	URIDINE-5'-DIPHOSPHATE C₉ H₁₄ N₂ O₁₂ P₂ XCCTYIAWTASOJW-XVFCMESISA-N		Interactions Focus chain J [auth A] Focus chain L [auth A] Focus chain N [auth B] Focus chain P [auth B] Focus chain R [auth C] Focus chain T [auth C] Focus chain V [auth D] Focus chain X [auth D] Interactions & Density Focus chain J [auth A] Focus chain L [auth A] Focus chain N [auth B] Focus chain P [auth B] Focus chain R [auth C] Focus chain T [auth C] Focus chain V [auth D] Focus chain X [auth D]
MN (Subject of Investigation/LOI) Query on MN Download Ideal Coordinates CCD File SDF format, chain I [auth A] SDF format, chain K [auth A] SDF format, chain M [auth B] SDF format, chain O [auth B] SDF format, chain Q [auth C] SDF format, chain S [auth C] SDF format, chain U [auth D] SDF format, chain W [auth D] MOL2 format, chain I [auth A] MOL2 format, chain K [auth A] MOL2 format, chain M [auth B] MOL2 format, chain O [auth B] MOL2 format, chain Q [auth C] MOL2 format, chain S [auth C] MOL2 format, chain U [auth D] MOL2 format, chain W [auth D] mmCIF format, chain I [auth A] mmCIF format, chain K [auth A] mmCIF format, chain M [auth B] mmCIF format, chain O [auth B] mmCIF format, chain Q [auth C] mmCIF format, chain S [auth C] mmCIF format, chain U [auth D] mmCIF format, chain W [auth D]	I [auth A] K [auth A] M [auth B] O [auth B] Q [auth C] I [auth A], K [auth A], M [auth B], O [auth B], Q [auth C], S [auth C], U [auth D], W [auth D]	MANGANESE (II) ION Mn WAEMQWOKJMHJLA-UHFFFAOYSA-N		Interactions Focus chain I [auth A] Focus chain K [auth A] Focus chain M [auth B] Focus chain O [auth B] Focus chain Q [auth C] Focus chain S [auth C] Focus chain U [auth D] Focus chain W [auth D] Interactions & Density Focus chain I [auth A] Focus chain K [auth A] Focus chain M [auth B] Focus chain O [auth B] Focus chain Q [auth C] Focus chain S [auth C] Focus chain U [auth D] Focus chain W [auth D]