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- PDB-9dh0: The Cryo-EM structure of recombinantly expressed hUGDH in complex... -

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Basic information

Entry
Database: PDB / ID: 9dh0
TitleThe Cryo-EM structure of recombinantly expressed hUGDH in complex with UDP-4-keto-xylose
ComponentsUDP-glucose 6-dehydrogenase
KeywordsOXIDOREDUCTASE/INHIBITOR / Human UDP-Glucose Dehydrogenase / UDP-4-keto-xylose / OXIDOREDUCTASE / OXIDOREDUCTASE-INHIBITOR complex
Function / homology
Function and homology information


Formation of the active cofactor, UDP-glucuronate / : / UDP-glucose 6-dehydrogenase activity / UDP-glucose 6-dehydrogenase / UDP-glucuronate biosynthetic process / glycosaminoglycan biosynthetic process / heparan sulfate proteoglycan biosynthetic process / gastrulation with mouth forming second / protein hexamerization / neuron development ...Formation of the active cofactor, UDP-glucuronate / : / UDP-glucose 6-dehydrogenase activity / UDP-glucose 6-dehydrogenase / UDP-glucuronate biosynthetic process / glycosaminoglycan biosynthetic process / heparan sulfate proteoglycan biosynthetic process / gastrulation with mouth forming second / protein hexamerization / neuron development / NAD binding / extracellular exosome / nucleoplasm / identical protein binding / nucleus / cytosol
Similarity search - Function
UDP-glucose 6-dehydrogenase, eukaryotic type / UDP-glucose/GDP-mannose dehydrogenase, N-terminal / UDP-glucose/GDP-mannose dehydrogenase, dimerisation / UDP-glucose/GDP-mannose dehydrogenase, C-terminal / UDP-glucose/GDP-mannose dehydrogenase / UDP-glucose/GDP-mannose dehydrogenase, C-terminal domain superfamily / UDP-glucose/GDP-mannose dehydrogenase family, central domain / UDP-glucose/GDP-mannose dehydrogenase family, UDP binding domain / UDP-glucose/GDP-mannose dehydrogenase family, NAD binding domain / UDP binding domain ...UDP-glucose 6-dehydrogenase, eukaryotic type / UDP-glucose/GDP-mannose dehydrogenase, N-terminal / UDP-glucose/GDP-mannose dehydrogenase, dimerisation / UDP-glucose/GDP-mannose dehydrogenase, C-terminal / UDP-glucose/GDP-mannose dehydrogenase / UDP-glucose/GDP-mannose dehydrogenase, C-terminal domain superfamily / UDP-glucose/GDP-mannose dehydrogenase family, central domain / UDP-glucose/GDP-mannose dehydrogenase family, UDP binding domain / UDP-glucose/GDP-mannose dehydrogenase family, NAD binding domain / UDP binding domain / 6-phosphogluconate dehydrogenase-like, C-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
: / UDP-glucose 6-dehydrogenase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.38 Å
AuthorsKadirvelraj, R. / Walsh Jr, R.M. / Wood, Z.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM114298 United States
Citation
Journal: Biochemistry / Year: 2025
Title: Cryo-EM Structure of Recombinantly Expressed hUGDH Unveils a Hidden, Alternative Allosteric Inhibitor.
Authors: John H O'Brien / Renuka Kadirvelraj / Po-Sen Tseng / Nolan Ross-Kemppinen / David Crich / Richard M Walsh / Zachary A Wood /
Abstract: Human UDP-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose into UDP-glucuronic acid, an essential substrate in the Phase II metabolism of drugs. hUGDH is a hexamer that exists in ...Human UDP-glucose dehydrogenase (hUGDH) catalyzes the oxidation of UDP-glucose into UDP-glucuronic acid, an essential substrate in the Phase II metabolism of drugs. hUGDH is a hexamer that exists in an equilibrium between an active (E) state and an inactive (E) state, with the latter being stabilized by the binding of the allosteric inhibitor UDP-xylose (UDP-Xyl). The allosteric transition between E and E is slow and can be observed as a lag in progress curves. Previous analysis of the lag suggested that unliganded hUGDH exists mainly as E, but two unique crystal forms suggest that the enzyme favors the E state. Resolving this discrepancy is necessary to fully understand the allosteric mechanism of hUGDH. Here, we used cryo-EM to show that recombinant hUGDH expressed in copurifies with UDP-4-keto-xylose (UX4O), which mimics the UDP-Xyl inhibitor and favors the E state. Cryo-EM studies show that removing UX4O from hUGDH shifts the ensemble to favor the E state. This shift is consistent with progress curve analysis, which shows the absence of a lag for unliganded hUGDH. Inhibition studies show that hUGDH has similar affinities for UDP-Xyl and UX4O. The discovery that UX4O inhibits allosteric hUGDH suggests that UX4O may be the physiologically relevant inhibitor of allosteric UGDHs in bacteria that do not make UDP-Xyl.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty ...Authors: Dorothee Liebschner / Pavel V Afonine / Matthew L Baker / Gábor Bunkóczi / Vincent B Chen / Tristan I Croll / Bradley Hintze / Li Wei Hung / Swati Jain / Airlie J McCoy / Nigel W Moriarty / Robert D Oeffner / Billy K Poon / Michael G Prisant / Randy J Read / Jane S Richardson / David C Richardson / Massimo D Sammito / Oleg V Sobolev / Duncan H Stockwell / Thomas C Terwilliger / Alexandre G Urzhumtsev / Lizbeth L Videau / Christopher J Williams / Paul D Adams /
Abstract: Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological ...Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.
History
DepositionSep 3, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 12, 2025Group: Author supporting evidence / Data collection / Category: em_admin / em_single_particle_entity / Item: _em_admin.last_update
Revision 1.1Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data collection / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_single_particle_entity / Data content type: EM metadata / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UDP-glucose 6-dehydrogenase
B: UDP-glucose 6-dehydrogenase
C: UDP-glucose 6-dehydrogenase
D: UDP-glucose 6-dehydrogenase
E: UDP-glucose 6-dehydrogenase
F: UDP-glucose 6-dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)332,70110
Polymers330,5646
Non-polymers2,1374
Water3,477193
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
UDP-glucose 6-dehydrogenase / UDP-Glc dehydrogenase / UDP-GlcDH / UDPGDH


Mass: 55093.938 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UGDH / Production host: Escherichia coli (E. coli) / References: UniProt: O60701, UDP-glucose 6-dehydrogenase
#2: Chemical
ChemComp-A1BCU / (2R,3R,4R)-3,4-dihydroxy-5-oxooxan-2-yl [(2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen diphosphate (non-preferred name) / Uridine-diphosphate-4-keto-D-xylose


Mass: 534.260 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H20N2O16P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 193 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human UDP-Glucose Dehydrogenase in complex with UDP-4-keto-xylose
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.055024 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2100 mMSodium ChlorideNaCl1
31 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 20 mM Hepes pH 7.5, 100 mM NaCl and 1 mM DTT.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 54.42 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13500

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2PHENIX1.21.1_5286model refinement
3SerialEMimage acquisition
10RELIONinitial Euler assignment
11cryoSPARCfinal Euler assignment
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4118676
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 3059917 / Symmetry type: POINT

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