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- PDB-8vh8: Crystal structure of heparosan synthase 2 from Pasteurella multoc... -

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Basic information

Entry
Database: PDB / ID: 8vh8
TitleCrystal structure of heparosan synthase 2 from Pasteurella multocida at 2.85 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: 2.85 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 1.0Sep 4, 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)259,34847
Polymers254,6574
Non-polymers4,69143
Water39622
1
A: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,7129
Polymers63,6641
Non-polymers1,0488
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,91113
Polymers63,6641
Non-polymers1,24712
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,91614
Polymers63,6641
Non-polymers1,25113
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
4
D: Heparosan synthase B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,80911
Polymers63,6641
Non-polymers1,14510
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)118.570, 118.570, 262.356
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number78
Space group name H-MP43
Space group name HallP4cw
Symmetry operation#1: x,y,z
#2: -y,x,z+3/4
#3: y,-x,z+1/4
#4: -x,-y,z+1/2

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Heparosan synthase B


Mass: 63664.188 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pasteurella multocida (bacteria) / Gene: hssB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2 / References: UniProt: Q5SGE1

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Non-polymers , 7 types, 65 molecules

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


Mass: 54.938 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mn
#3: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: UDP*YM
#4: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na
#5: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Cl
#6: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#7: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 22 / 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: 3.62 Å3/Da / Density % sol: 66.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: Protein: 15mg/ml PmHS2(98-644) in 25mM Tris pH 7.5, 125mM NaCl, 5mM MnCl2, 5mM UDPGlcNAc, UDPGlcA well solution: .1M imidazole/MDES pH6.5, 60mM (MgCl2 and CaCl2)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 19-ID / Wavelength: 0.979497 Å
DetectorType: DECTRIS EIGER2 XE 9M / Detector: PIXEL / Date: Jun 22, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979497 Å / Relative weight: 1
ReflectionResolution: 2.85→28.76 Å / Num. obs: 83838 / % possible obs: 98.72 % / Redundancy: 5.3 % / Biso Wilson estimate: 72.75 Å2 / CC1/2: 0.999 / Rpim(I) all: 0.04076 / Rrim(I) all: 0.09485 / Rsym value: 0.08541 / Net I/σ(I): 9.67
Reflection shellResolution: 2.852→2.954 Å / Mean I/σ(I) obs: 1.15 / Num. unique obs: 8067 / CC1/2: 0.657 / CC star: 0.89 / Rpim(I) all: 0.4425 / Rrim(I) all: 1.052 / Rsym value: 0.953

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.85→28.76 Å / SU ML: 0.3689 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.548
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2228 1979 2.39 %
Rwork0.1981 80885 -
obs0.1987 82864 98.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 81.3 Å2
Refinement stepCycle: LAST / Resolution: 2.85→28.76 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms16422 0 247 22 16691
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.006417013
X-RAY DIFFRACTIONf_angle_d0.838323150
X-RAY DIFFRACTIONf_chiral_restr0.05112644
X-RAY DIFFRACTIONf_plane_restr0.00652949
X-RAY DIFFRACTIONf_dihedral_angle_d12.09896033
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.85-2.920.30951420.30635554X-RAY DIFFRACTION96.25
2.92-30.35181410.29365698X-RAY DIFFRACTION97.11
3-3.090.29881370.27715689X-RAY DIFFRACTION97.62
3.09-3.190.30811410.28095735X-RAY DIFFRACTION98.39
3.19-3.30.30121360.25685803X-RAY DIFFRACTION98.47
3.3-3.440.2531390.24145751X-RAY DIFFRACTION99.24
3.44-3.590.26081390.22695830X-RAY DIFFRACTION99.5
3.59-3.780.24791400.20935811X-RAY DIFFRACTION99.85
3.78-4.020.21611490.19095858X-RAY DIFFRACTION99.82
4.02-4.330.20921380.16985837X-RAY DIFFRACTION99.93
4.33-4.760.16261440.15335831X-RAY DIFFRACTION99.88
4.76-5.440.17191450.16615870X-RAY DIFFRACTION99.97
5.45-6.840.22031460.20575838X-RAY DIFFRACTION99.83
6.84-28.760.21420.16375780X-RAY DIFFRACTION97.51
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)
10.795519367185-0.630001763438-0.07896930278432.005164636320.3789230966563.102005289550.07291286283520.221865025648-0.1003257988640.129461897742-0.0676583031957-0.0825120155101-0.02766068359550.06398341378130.00734257659010.560644984395-0.0481706384844-0.04999343397560.557590318179-0.02220874912270.67473745747431.85810466341.4338490998-33.0689327495
20.885902683436-0.120493240421-0.08170167741452.48617478870.7553505477772.423682405740.02514793007730.133390872701-0.003786441556170.44786957592-0.1721006763440.187119087697-0.212390581485-0.2897429362510.12590392870.648541485688-0.05539498412240.01509482379690.493563885143-0.02708418445570.67258508128321.935243076646.296428548-22.6692850688
31.44514091172-0.016494473675-0.3374996167520.601470601363-0.5503773250952.26493172206-0.20408509344-0.6877382506680.1641942233861.147243947160.109278033524-0.269858217465-0.1351555716990.1256902661780.1170687880461.548637788510.0219050785072-0.2940107335760.780480878867-0.005218452353820.89644056073441.33060759835.63336815393.61628831583
40.666083612959-0.2611629918430.4231386767251.318505443490.2224497158191.00464035124-0.0423055702752-0.135939259763-0.3294241094180.6418634782110.0924809295059-0.2777259941250.3127248332750.253923243126-0.05048352069621.267424971010.0242674499493-0.1360524796310.6172075581130.0872334652150.88279973236139.478902721317.6096797233-4.85557596647
51.54917937549-0.554166362995-0.4661682166841.02999201317-0.7234146494873.183104065240.02436769323590.1887613039080.1336434658860.0482221552967-0.0292197981825-0.1172454750780.0683312566002-0.04461241650510.01592525359870.60043514631-0.0783922376447-0.07514925651590.4372492961550.0215266505310.49231782863-32.180728713178.0608318006-36.2802956041
61.16046749650.006762560772410.1307237401391.83458885128-0.425711133591.467040383280.01815869003060.04093281546680.07076583786420.244914019014-0.0815449880864-0.1625336602280.2452655702870.2916585620660.08383439953040.697842084424-0.0627043208757-0.07668416296690.458740908470.05523995396250.567341555895-22.050959793273.4352552567-25.9083450681
71.77380132878-0.355136786389-0.2448853375951.87848429531-0.7444855172561.09578543663-0.00884991192944-0.328244027520.036759264810.5284485660990.08330580751920.4084951686350.00365743208972-0.179426837066-0.05756880204040.947843622981-0.08634795521030.05040996885250.516200978767-0.06880318682110.630384998364-40.663253552894.1481001554-4.68739944759
80.634765541730.2927219320540.4910009580421.6894055545-1.115493426121.05079696989-0.0389391291348-0.2018387636350.313733421980.08436916487870.1343727172820.452063792204-0.04471236357820.0281656698007-0.1709688071521.00977179353-0.003572962623440.03401689608990.694733717593-0.07377401362121.00957078731-41.3101952452109.044889381-7.74568948332
90.9145021215770.158632750981-0.4236616039120.5673911748670.1904969363093.057711760430.0478903218612-0.424357080239-0.1536365810390.180750899503-0.0291963394893-0.02166659730410.548016744141-0.2344719653970.08424911515070.865124626768-0.0330158133293-0.05769023596440.5801290462710.07071101772970.68957815240330.180234528874.793463667816.9157074301
100.9762465761760.299197086906-0.2339398458070.9455144984721.237625857962.927395889510.000331139201731-0.1254965228070.00400891935213-0.200066463196-0.00318099956771-0.0669083998995-0.03789668195950.0990813120431-0.002875990247240.7530277679980.0242383609016-0.06365657974160.4364539391360.05596642033780.69518950216535.816259865883.11609874497.66358979823
111.700930905520.04165757953110.07467569239251.298959387521.321108230093.000397280410.0665569720204-0.0979665302792-0.0819719675829-0.000489259204919-0.1646541483910.08239360739820.433571802427-0.1334032929440.1252789564480.822490880305-0.0473797423631-0.03260935505010.4525324472330.05578910795710.73162655167723.907323500370.61849817197.20556458157
121.58802021052-0.1563229088040.04450515100661.128671227220.4379031817952.26501955894-0.0837371380196-0.2768612915550.0257760911787-0.126067333415-0.1187799717690.2040311803220.0546018586376-0.8515063122580.2784541215350.74570386304-0.00560201583237-0.02796798660620.82038356783-0.02778795218750.88199623335313.037096304680.61174580496.28876506453
131.347733650680.956570002515-0.1376616318912.80352100316-0.5703569218611.52627302118-0.1763493367740.271759625166-0.11076849814-0.7064977858510.0106844414535-0.423615853470.137496332047-0.1002204335390.1036363856530.9974494680960.05831848425570.01794994333940.49753477592-0.003583560449530.7859560379730.935582472283.0137925471-15.8867434233
141.826829646890.49568568788-0.9012043391121.379924509650.4827151774591.444760904720.1424119266880.359611037761-0.113890902608-0.648097124054-0.146888501758-0.237890453351-0.7093868129950.1348463442920.03211671458541.33417070533-0.1590729871360.1394547515410.8416422504890.01988686044160.95519370461541.91746250987.384317726-29.1691981125
151.405054393-0.37997204443-0.4917991910831.997740829420.4034003361671.255614380340.08390311228320.3868435933160.13213910731-0.366657781384-0.0012125875609-0.324345004059-0.2157212523140.117449140043-0.06842523649931.08555702553-0.006750019130820.06493002622290.6351908297260.1147320543520.87191499649238.2704015505101.188771864-17.9074509455
160.4748563549821.020876901840.1881787279291.9184005750.8152282192051.43694935052-0.03051701049310.3025590217860.35644646267-0.6030324883760.265880981416-0.1826690911-0.5346897389590.128601654072-0.1519597418491.17493143375-0.1074995227190.02770449017760.6432851712860.1280565111350.96179076008639.9397317991109.774763939-17.386897432
171.39141969255-0.0328714645922-0.3213824351561.18598275882-0.2494892248232.230599342670.124344006759-0.394247946270.00823526339950.308811107028-0.1066705899420.0947494345878-0.4734106992120.245066827328-0.01647809016450.72473889794-0.0556913360246-0.0008724291791810.624133488590.06728585945530.457133988957-29.534202115145.04059767813.7466501511
180.8617341453840.021606267474-0.07125013922880.973325158773-0.2041287856373.62141650987-0.00158340624878-0.208189303458-0.1303935096880.07468023659160.01150062927910.0724769104256-0.100203773261-0.1242651786550.01015170569210.523242733770.0313185271226-0.01631760869160.545532268730.05734824820730.579973646392-35.274132484436.27376169754.40799388183
191.77930916131-0.0759627283224-0.695278780531.55776032059-1.037536219953.493421152560.00960224033426-0.2413167120240.1105271747430.121927154021-0.0986572431546-0.185704801444-0.5726213700890.3206187696780.08248085139560.660528137001-0.0422383082623-0.08224371286660.5256897062180.03865204203530.573830782182-23.427622074748.83730835843.84765909014
201.525433173270.08494094602310.04333250876190.8983167310680.1812130769142.02627943140.00764378441284-0.123126916818-0.2537115612110.0119102676716-0.0676818844876-0.425922506132-0.1149425125420.7130140161580.2008853690530.631992774267-0.0357182215389-0.07669664583120.8302958904310.06948428781050.785227346917-12.640839087838.8380935392.73241446338
210.730771256920.7754733261-0.3801428402791.929627094191.007242375151.42499382143-0.04900220536620.09066987620960.0339506931513-0.3701611147340.0635996221869-0.0119443223042-0.226877093490.08168004886190.01450608727150.690742220377-0.00318550081098-0.03406844319780.5007005900630.07508357961860.618203775094-25.882012241337.0006270777-18.5675657133
222.11429665419-0.06925783396370.6198435587381.86215823937-0.8553612143941.99634031761-0.2399749287720.1295633130240.35203060477-0.36157816680.1106188087130.21489641232-0.411603756626-0.2963314631640.1747230654430.8261695445040.0730111429805-0.1329812666180.7512400839440.03430014544350.706668217586-43.049451544533.2249571226-30.7706216804
232.3811705883-0.08779307588360.4739899734292.8322926230.3148759563642.624375849670.02707811812810.284792117731-0.284817936099-0.202287507268-0.08730744184020.1736499515140.377832173318-0.1012197574330.06576298722520.6644743941730.00397744559139-0.0525488974940.509354415717-0.0512020563050.660362913633-38.154689706717.0259644859-22.4472438488
240.6964401958881.09954418138-0.4132220711642.06468612753-0.5054869800272.10838873916-0.02010801065960.162005419481-0.261457975066-0.212441980110.005410804826570.097027937760.364991318417-0.2514955683970.07407901499920.723701180261-0.0228628453208-0.0557771855130.634918083703-0.09070537577230.752360976654-41.00563686019.80045681047-19.0168300104
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 101 through 223 )AA101 - 2231 - 118
22chain 'A' and (resid 224 through 414 )AA224 - 414119 - 309
33chain 'A' and (resid 415 through 487 )AA415 - 487310 - 382
44chain 'A' and (resid 488 through 644 )AA488 - 644383 - 525
55chain 'B' and (resid 101 through 223 )BH101 - 2231 - 119
66chain 'B' and (resid 224 through 414 )BH224 - 414120 - 310
77chain 'B' and (resid 415 through 595 )BH415 - 595311 - 491
88chain 'B' and (resid 596 through 644 )BH596 - 644492 - 529
99chain 'C' and (resid 101 through 141 )CP101 - 1411 - 37
1010chain 'C' and (resid 142 through 223 )CP142 - 22338 - 119
1111chain 'C' and (resid 224 through 313 )CP224 - 313120 - 209
1212chain 'C' and (resid 314 through 358 )CP314 - 358210 - 254
1313chain 'C' and (resid 359 through 414 )CP359 - 414255 - 310
1414chain 'C' and (resid 415 through 468 )CP415 - 468311 - 364
1515chain 'C' and (resid 469 through 567 )CP469 - 567365 - 457
1616chain 'C' and (resid 568 through 644 )CP568 - 644458 - 521
1717chain 'D' and (resid 101 through 141 )DX101 - 1411 - 38
1818chain 'D' and (resid 142 through 223 )DX142 - 22339 - 120
1919chain 'D' and (resid 224 through 313 )DX224 - 313121 - 210
2020chain 'D' and (resid 314 through 358 )DX314 - 358211 - 255
2121chain 'D' and (resid 359 through 400 )DX359 - 400256 - 297
2222chain 'D' and (resid 401 through 468 )DX401 - 468298 - 365
2323chain 'D' and (resid 469 through 585 )DX469 - 585366 - 477
2424chain 'D' and (resid 586 through 644 )DX586 - 644478 - 524

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