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- PDB-8pt4: beta-Ureidopropionase tetramer -

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Basic information

Entry
Database: PDB / ID: 8pt4
Titlebeta-Ureidopropionase tetramer
ComponentsBeta-ureidopropionase
KeywordsBIOSYNTHETIC PROTEIN / Pyrimidine degradation / drug metabolism
Function / homology
Function and homology information


pyrimidine nucleoside catabolic process / beta-alanine biosynthetic process via 3-ureidopropionate / beta-ureidopropionase activity / beta-ureidopropionase / CMP catabolic process / dCMP catabolic process / UMP catabolic process / dUMP catabolic process / Pyrimidine catabolism / liver development ...pyrimidine nucleoside catabolic process / beta-alanine biosynthetic process via 3-ureidopropionate / beta-ureidopropionase activity / beta-ureidopropionase / CMP catabolic process / dCMP catabolic process / UMP catabolic process / dUMP catabolic process / Pyrimidine catabolism / liver development / protein homooligomerization / protein homotetramerization / in utero embryonic development / protein homodimerization activity / extracellular exosome / cytosol
Similarity search - Function
: / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Beta-ureidopropionase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsCederfelt, D. / Dobritzsch, D.
Funding support Sweden, 1items
OrganizationGrant numberCountry
Carl Trygger FoundationCTS18:84 Sweden
CitationJournal: Biomolecules / Year: 2023
Title: The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.
Authors: Daniela Cederfelt / Dilip Badgujar / Ayan Au Musse / Bernhard Lohkamp / U Helena Danielson / Doreen Dobritzsch /
Abstract: The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the ...The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.
History
DepositionJul 13, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-ureidopropionase
B: Beta-ureidopropionase
C: Beta-ureidopropionase
D: Beta-ureidopropionase


Theoretical massNumber of molelcules
Total (without water)172,8764
Polymers172,8764
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Beta-ureidopropionase / BUP-1 / Beta-alanine synthase / N-carbamoyl-beta-alanine amidohydrolase


Mass: 43218.965 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UPB1, BUP1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UBR1, beta-ureidopropionase

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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: beta-Ureidopropionase tetramer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.43 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5
Details: 100 mM sodium acetate, 50 mM sodium chloride, pH 5.0
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium acetateC2H3NaO21
250 mMSodium chlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 600 nm
Image recordingElectron dose: 43.661 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 4.2.1 / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 1/2 BIT CUT-OFF / Num. of particles: 169949 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211726
ELECTRON MICROSCOPYf_angle_d0.55115911
ELECTRON MICROSCOPYf_dihedral_angle_d11.8764291
ELECTRON MICROSCOPYf_chiral_restr0.0381702
ELECTRON MICROSCOPYf_plane_restr0.0042095

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