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- PDB-8vh7: Crystal structure of heparosan synthase 2 from Pasteurella multoc... -

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Basic information

Entry
Database: PDB / ID: 8vh7
TitleCrystal structure of heparosan synthase 2 from Pasteurella multocida at 1.98 A
ComponentsHeparosan synthase B
KeywordsTRANSFERASE / glycosyltransferase / heparosan / chemoenzymatic synthesis / heparan sulfate
Function / homologyGlycosyltransferase 2-like / Glycosyl transferase family 2 / Nucleotide-diphospho-sugar transferases / nucleotide binding / metal ion binding / : / URIDINE-5'-DIPHOSPHATE / Heparosan synthase B
Function and homology information
Biological speciesPasteurella multocida (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.982 Å
AuthorsPedersen, L.C. / Liu, J. / Stancanelli, E. / Krahn, J.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)ZIC-ES102645 United States
Department of Health & Human Services (HHS)R44GM142304 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)1R01HL094463-09 United States
CitationJournal: ACS Catal / Year: 2024
Title: Structural and Functional Analysis of Heparosan Synthase 2 from (PmHS2) to Improve the Synthesis of Heparin.
Authors: Eduardo Stancanelli / Juno A Krahn / Elizabeth Viverette / Robert Dutcher / Vijayakanth Pagadala / Mario J Borgnia / Jian Liu / Lars C Pedersen /
Abstract: Heparin is a widely used drug to treat thrombotic disorders in hospitals. Heparosan synthase 2 from (PmHS2) is a key enzyme used for the chemoenzymatic synthesis of heparin oligosaccharides. It has ...Heparin is a widely used drug to treat thrombotic disorders in hospitals. Heparosan synthase 2 from (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.
History
DepositionDec 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Database references / Structure summary / Category: citation / pdbx_entry_details
Item: _citation.pdbx_database_id_PubMed / _citation.title / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Heparosan synthase B
B: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,00226
Polymers127,3282
Non-polymers2,67324
Water15,349852
1
A: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,96213
Polymers63,6641
Non-polymers1,29812
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)65,04013
Polymers63,6641
Non-polymers1,37612
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)93.178, 163.177, 84.273
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212
Space group name HallP22ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x+1/2,y+1/2,-z
#4: -x,-y,z
Components on special symmetry positions
IDModelComponents
11B-952-

HOH

21B-1151-

HOH

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Heparosan synthase B


Mass: 63664.188 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pasteurella multocida (bacteria) / Gene: hssB / Production host: Escherichia coli (E. coli) / References: UniProt: Q5SGE1

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Non-polymers , 5 types, 876 molecules

#2: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 852 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.52 Å3/Da / Density % sol: 51.11 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: ML: Morpheus 2-26 0.1M carboxylic Acids, 0.1M Buffer system 1 pH 6.5, 30% Pmix2 EDO_P8K 5mM UDP, 5mM MnCl2, 5mM NS-tetrmer

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.979342 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: Jun 14, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979342 Å / Relative weight: 1
ReflectionResolution: 1.982→45.7 Å / Num. obs: 89657 / % possible obs: 99.61 % / Redundancy: 7.9 % / Biso Wilson estimate: 24.22 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.1601 / Rpim(I) all: 0.05974 / Rrim(I) all: 0.1711 / Net I/σ(I): 8.36
Reflection shellResolution: 1.982→2.053 Å / Redundancy: 7.9 % / Rmerge(I) obs: 1.023 / Mean I/σ(I) obs: 1.88 / Num. unique obs: 8728 / CC1/2: 0.84 / Rpim(I) all: 0.3812 / Rrim(I) all: 1.093 / % possible all: 98.48

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
autoPROCdata reduction
autoPROCdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.982→45.7 Å / SU ML: 0.2391 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 29.6026
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.249 2048 2.29 %
Rwork0.208 87419 -
obs0.2089 89467 99.62 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 30.65 Å2
Refinement stepCycle: LAST / Resolution: 1.982→45.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8262 0 156 852 9270
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00698602
X-RAY DIFFRACTIONf_angle_d0.892211666
X-RAY DIFFRACTIONf_chiral_restr0.05341325
X-RAY DIFFRACTIONf_plane_restr0.00811475
X-RAY DIFFRACTIONf_dihedral_angle_d13.10633097
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.982-2.030.33261320.29395648X-RAY DIFFRACTION97.75
2.03-2.080.27481350.24745753X-RAY DIFFRACTION99.95
2.08-2.140.31761340.22925784X-RAY DIFFRACTION99.93
2.14-2.20.26741360.21275779X-RAY DIFFRACTION99.9
2.2-2.270.28041360.20975802X-RAY DIFFRACTION99.92
2.27-2.350.26211360.20455774X-RAY DIFFRACTION99.86
2.35-2.440.2691360.19285818X-RAY DIFFRACTION99.93
2.44-2.560.23451360.18795800X-RAY DIFFRACTION99.9
2.56-2.690.23091360.18455807X-RAY DIFFRACTION99.85
2.69-2.860.27031370.18955833X-RAY DIFFRACTION99.85
2.86-3.080.2361370.19065848X-RAY DIFFRACTION99.83
3.08-3.390.2491360.19845842X-RAY DIFFRACTION99.62
3.39-3.880.20331380.18945898X-RAY DIFFRACTION99.88
3.88-4.890.21441390.18345936X-RAY DIFFRACTION99.82
4.89-45.70.27751440.26896097X-RAY DIFFRACTION98.39
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.316796800426-0.102900670571-0.1167152675780.896425958155-0.01905172363990.6821325708370.005283444654940.01120854323970.0470393617707-0.05618868480080.001792214926290.0233914305673-0.0356209639720.00833998802525-0.001874068564830.19475523373-0.0207257759858-0.003745733936940.1958950830030.01162791326640.189504999888-26.302441955420.6262923853-18.7487648452
20.363259281122-0.241854653503-0.05380910473030.8915890054080.2056222060280.604255844788-0.0835201364918-0.02284820025370.04256919875690.07366999895890.06114868844570.017855137575-0.04241795776890.05562813270560.005371710842570.188199653004-0.03633214675150.007581207677430.2589270459750.03192503530620.19259825841-29.00150283796.02568683596-7.31410662062
31.308664510090.267926135762-0.731979376421.34254674783-0.2898187930322.71473997587-0.0663863316386-0.109123376191-0.2497197397580.0490077688091-0.0439971308384-0.190409976350.2362158108420.4641928179220.04444484315090.165365094420.05266859442040.0158407070130.2177497085510.004696708437050.205465317751-16.4373402133-16.5180804682-15.3338568957
42.66297745919-0.630977428059-0.6150648214452.239067942980.740882230132.87722170009-0.4062592719280.625740126974-0.6220478283350.07700593196640.07771429337810.1593553605810.5682831940820.04530088385690.2641778452590.4870456159270.0105036194750.02796864939270.320639208652-0.03966442489790.281274113695-21.1168257646-25.4120990503-27.3950192276
50.531016376772-0.0683575884711-0.1675705601071.148487468070.1674705816241.03616177363-0.00972962804504-0.0502210764548-0.06374287691660.1501716554330.0006700943892730.06316889518870.142541174015-0.05130294540440.008655329722930.1982821925910.023531014963-0.002821548045840.167363640013-0.008056989363220.181976891102-26.2724648488-20.6215186483-55.4811589651
60.5405897080960.506299600035-0.2739077910651.38776437632-0.1412767222690.580045284365-0.0281445130038-0.0584294549803-0.0143555929553-0.1190030142440.009442381326290.0797055971450.06809173987860.0728971206098-0.01028964059130.1701657835410.0431911005101-0.004934040470430.2285593836880.0002343397067340.165265361832-29.1212614195-5.96477135307-66.8942224083
71.24794081601-0.2857620352930.7122879587441.06629492591-0.1597558555133.52343764398-0.06814159798860.1350807214530.188032808168-0.0300803682953-0.0157810285221-0.148041763809-0.3233204730430.5612856269630.01255590984770.171574633848-0.0567894024432-0.01447020272830.2361598601290.002665036680470.201038721665-15.874263358117.3005584001-57.9782453743
86.822915608221.378112362.012302838732.319793623240.5865508624343.83763849829-0.266201841132-0.5830795582610.685579545735-0.04529109905420.01117067285990.281881208925-0.897618292066-0.6641247794050.1510662353620.4545513568070.0599911221394-0.003495070260040.300415943714-0.07679070955980.322873217912-30.358671235625.3217798456-45.5876289262
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 102 through 323 )AA102 - 3231 - 217
22chain 'A' and (resid 324 through 414 )AA324 - 414218 - 308
33chain 'A' and (resid 415 through 595 )AA415 - 595309 - 482
44chain 'A' and (resid 596 through 644 )AA596 - 644483 - 522
55chain 'B' and (resid 101 through 323 )BH101 - 3231 - 218
66chain 'B' and (resid 324 through 414 )BH324 - 414219 - 309
77chain 'B' and (resid 415 through 621 )BH415 - 621310 - 499
88chain 'B' and (resid 622 through 644 )BH622 - 644500 - 522

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