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- PDB-8pw3: Mycobacterium smegmatis inosine monophosphate dehydrogenase (IMPD... -

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Basic information

Entry
Database: PDB / ID: 8pw3
TitleMycobacterium smegmatis inosine monophosphate dehydrogenase (IMPDH) apo form
ComponentsInosine-5'-monophosphate dehydrogenase
KeywordsOXIDOREDUCTASE / Octamer / Apo enzyme / Purine metabolism / IMPDH
Function / homology
Function and homology information


IMP dehydrogenase activity / IMP dehydrogenase / GMP biosynthetic process / GTP biosynthetic process / nucleotide binding / metal ion binding
Similarity search - Function
Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / GMP reductase, conserved site / IMP dehydrogenase / GMP reductase signature. / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / IMP dehydrogenase / GMP reductase domain / Domain in cystathionine beta-synthase and other proteins. / CBS domain superfamily / CBS domain / CBS domain ...Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / GMP reductase, conserved site / IMP dehydrogenase / GMP reductase signature. / IMP dehydrogenase/GMP reductase / IMP dehydrogenase / GMP reductase domain / IMP dehydrogenase / GMP reductase domain / Domain in cystathionine beta-synthase and other proteins. / CBS domain superfamily / CBS domain / CBS domain / CBS domain profile. / Aldolase-type TIM barrel
Similarity search - Domain/homology
Inosine-5'-monophosphate dehydrogenase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.507 Å
AuthorsBulvas, O. / Kouba, T. / Pichova, I.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Union (EU)LX22NPO5103European Union
CitationJournal: Nat Commun / Year: 2024
Title: Deciphering the allosteric regulation of mycobacterial inosine-5'-monophosphate dehydrogenase.
Authors: Ondřej Bulvas / Zdeněk Knejzlík / Jakub Sýs / Anatolij Filimoněnko / Monika Čížková / Kamila Clarová / Dominik Rejman / Tomáš Kouba / Iva Pichová /
Abstract: Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the ...Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.
History
DepositionJul 19, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Inosine-5'-monophosphate dehydrogenase
B: Inosine-5'-monophosphate dehydrogenase
C: Inosine-5'-monophosphate dehydrogenase
D: Inosine-5'-monophosphate dehydrogenase
E: Inosine-5'-monophosphate dehydrogenase
F: Inosine-5'-monophosphate dehydrogenase
G: Inosine-5'-monophosphate dehydrogenase
H: Inosine-5'-monophosphate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)427,1128
Polymers427,1128
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Inosine-5'-monophosphate dehydrogenase / IMP dehydrogenase / IMPD / IMPDH


Mass: 53388.988 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: MC2 155 / Gene: guaB, MSMEG_1602 / Plasmid: pRSFDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): LOBSTR- -RIL / References: UniProt: A0QSU3, IMP dehydrogenase

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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: Octameric assembly of inosine monophosphate dehydrogenase
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.426658 MDa / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: LOBSTR-BL21(DE3)-RIL / Plasmid: pRSFDuet-1
Buffer solutionpH: 7.5
Details: 50 mM HEPES (pH 7.5), 200 mM KCl, 5 mM DTT,2 mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acidHEPES1
2500 mMPotassium chlorideKCl1
35 mMDithiothreitolDTT1
42 mMMagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 magnification: 165000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7UCSF ChimeraX1.6.1model fitting
13PHENIX1.20.1model refinement
14Coot0.9.8 ELmodel refinement
15ISOLDE1.4model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 5882510
3D reconstructionResolution: 2.507 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 550995 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 36.96 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC coefficient
Details: Initial fitting was done in UCSF ChimeraX. Model refinement was done by iterative cycles of manual fitting with Coot and ISOLDE and automated fitting with phenix.real_space_refine.
Atomic model buildingAccession code: A0QSU3 / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 36.96 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002420032
ELECTRON MICROSCOPYf_angle_d0.48427208
ELECTRON MICROSCOPYf_chiral_restr0.0423344
ELECTRON MICROSCOPYf_plane_restr0.00383544
ELECTRON MICROSCOPYf_dihedral_angle_d4.63732904

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