8VH7 | pdb_00008vh7

Crystal structure of heparosan synthase 2 from Pasteurella multocida at 1.98 A

PDB DOI: https://doi.org/10.2210/pdb8VH7/pdb

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

Deposited: 2023-12-31 Released: 2024-07-24
Deposition Author(s): Pedersen, L.C., Liu, J., Stancanelli, E., Krahn, J.M.
Funding Organization(s): National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS), Department of Health & Human Services (HHS), National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.98 Å
R-Value Free:
0.249 (Depositor), 0.250 (DCC)
R-Value Work:
0.208 (Depositor), 0.210 (DCC)
R-Value Observed:
0.209 (Depositor)

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

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: 130 kDa
Atom Count: 9,307
Modelled Residue Count: 1,044
Deposited Residue Count: 1,104
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	552	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

Small Molecules

Ligands 4 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 D [auth A] SDF format, chain F [auth A] SDF format, chain P [auth B] SDF format, chain R [auth B] MOL2 format, chain D [auth A] MOL2 format, chain F [auth A] MOL2 format, chain P [auth B] MOL2 format, chain R [auth B] mmCIF format, chain D [auth A] mmCIF format, chain F [auth A] mmCIF format, chain P [auth B] mmCIF format, chain R [auth B]	D [auth A], F [auth A], P [auth B], R [auth B]	URIDINE-5'-DIPHOSPHATE C₉ H₁₄ N₂ O₁₂ P₂ XCCTYIAWTASOJW-XVFCMESISA-N		Interactions Focus chain D [auth A] Focus chain F [auth A] Focus chain P [auth B] Focus chain R [auth B] Interactions & Density Focus chain D [auth A] Focus chain F [auth A] Focus chain P [auth B] Focus chain R [auth B]
EDO Query on EDO Download Ideal Coordinates CCD File SDF format, chain G [auth A] SDF format, chain H [auth A] SDF format, chain L [auth A] SDF format, chain M [auth A] SDF format, chain N [auth A] SDF format, chain T [auth B] SDF format, chain U [auth B] SDF format, chain V [auth B] SDF format, chain W [auth B] SDF format, chain X [auth B] SDF format, chain Y [auth B] SDF format, chain Z [auth B] MOL2 format, chain G [auth A] MOL2 format, chain H [auth A] MOL2 format, chain L [auth A] MOL2 format, chain M [auth A] MOL2 format, chain N [auth A] MOL2 format, chain T [auth B] MOL2 format, chain U [auth B] MOL2 format, chain V [auth B] MOL2 format, chain W [auth B] MOL2 format, chain X [auth B] MOL2 format, chain Y [auth B] MOL2 format, chain Z [auth B] mmCIF format, chain G [auth A] mmCIF format, chain H [auth A] mmCIF format, chain L [auth A] mmCIF format, chain M [auth A] mmCIF format, chain N [auth A] mmCIF format, chain T [auth B] mmCIF format, chain U [auth B] mmCIF format, chain V [auth B] mmCIF format, chain W [auth B] mmCIF format, chain X [auth B] mmCIF format, chain Y [auth B] mmCIF format, chain Z [auth B]	G [auth A] H [auth A] L [auth A] M [auth A] N [auth A] G [auth A], H [auth A], L [auth A], M [auth A], N [auth A], T [auth B], U [auth B], V [auth B], W [auth B], X [auth B], Y [auth B], Z [auth B]	1,2-ETHANEDIOL C₂ H₆ O₂ LYCAIKOWRPUZTN-UHFFFAOYSA-N		Interactions Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth A] Focus chain M [auth A] Focus chain N [auth A] Focus chain T [auth B] Focus chain U [auth B] Focus chain V [auth B] Focus chain W [auth B] Focus chain X [auth B] Focus chain Y [auth B] Focus chain Z [auth B] Interactions & Density Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth A] Focus chain M [auth A] Focus chain N [auth A] Focus chain T [auth B] Focus chain U [auth B] Focus chain V [auth B] Focus chain W [auth B] Focus chain X [auth B] Focus chain Y [auth B] Focus chain Z [auth B]
MN (Subject of Investigation/LOI) Query on MN Download Ideal Coordinates CCD File SDF format, chain C [auth A] SDF format, chain E [auth A] SDF format, chain O [auth B] SDF format, chain Q [auth B] MOL2 format, chain C [auth A] MOL2 format, chain E [auth A] MOL2 format, chain O [auth B] MOL2 format, chain Q [auth B] mmCIF format, chain C [auth A] mmCIF format, chain E [auth A] mmCIF format, chain O [auth B] mmCIF format, chain Q [auth B]	C [auth A], E [auth A], O [auth B], Q [auth B]	MANGANESE (II) ION Mn WAEMQWOKJMHJLA-UHFFFAOYSA-N		Interactions Focus chain C [auth A] Focus chain E [auth A] Focus chain O [auth B] Focus chain Q [auth B] Interactions & Density Focus chain C [auth A] Focus chain E [auth A] Focus chain O [auth B] Focus chain Q [auth B]
NA Query on NA Download Ideal Coordinates CCD File SDF format, chain I [auth A] SDF format, chain J [auth A] SDF format, chain K [auth A] SDF format, chain S [auth B] MOL2 format, chain I [auth A] MOL2 format, chain J [auth A] MOL2 format, chain K [auth A] MOL2 format, chain S [auth B] mmCIF format, chain I [auth A] mmCIF format, chain J [auth A] mmCIF format, chain K [auth A] mmCIF format, chain S [auth B]	I [auth A], J [auth A], K [auth A], S [auth B]	SODIUM ION Na FKNQFGJONOIPTF-UHFFFAOYSA-N		Interactions Focus chain I [auth A] Focus chain J [auth A] Focus chain K [auth A] Focus chain S [auth B] Interactions & Density Focus chain I [auth A] Focus chain J [auth A] Focus chain K [auth A] Focus chain S [auth B]

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 1.98 Å
R-Value Free: 0.249 (Depositor), 0.250 (DCC)
R-Value Work: 0.208 (Depositor), 0.210 (DCC)
R-Value Observed: 0.209 (Depositor)

Space Group: P 2₁ 2₁ 2

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 93.178	α = 90
b = 163.177	β = 90
c = 84.273	γ = 90

Software Package:

Software Name	Purpose
PHENIX	refinement
autoPROC	data reduction
autoPROC	data scaling
PHASER	phasing

Structure Validation

View Full Validation Report

Ligand Structure Quality Assessment

Entry History & Funding Information

Deposition Data

Released Date: 2024-07-24

Deposition Author(s):

Funding Organization	Location	Grant Number
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)	United States	ZIC-ES102645
Department of Health & Human Services (HHS)	United States	R44GM142304
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)	United States	1R01HL094463-09