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- PDB-7e4x: Structure of Enolase from Mycobacterium tuberculosis -

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Basic information

Entry
Database: PDB / ID: 7e4x
TitleStructure of Enolase from Mycobacterium tuberculosis
ComponentsEnolase
KeywordsMETAL BINDING PROTEIN
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / glycolytic process / cell surface / magnesium ion binding / extracellular region
Similarity search - Function
Enolase / Enolase, conserved site / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / Enolase signature. / Enolase, C-terminal TIM barrel domain / Enolase, N-terminal domain / Enolase-like, N-terminal / Enolase-like, C-terminal domain superfamily
Similarity search - Domain/homology
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsBose, S. / Vinothkumar, K.R.
Funding support India, 2items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB) India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: IUCrJ / Year: 2023
Title: Structural snapshots of Mycobacterium tuberculosis enolase reveal dual mode of 2PG binding and its implication in enzyme catalysis.
Authors: Mohammed Ahmad / Bhavya Jha / Sucharita Bose / Satish Tiwari / Abhisek Dwivedy / Deepshikha Kar / Ravikant Pal / Richard Mariadasse / Tanya Parish / Jeyaraman Jeyakanthan / Kutti R ...Authors: Mohammed Ahmad / Bhavya Jha / Sucharita Bose / Satish Tiwari / Abhisek Dwivedy / Deepshikha Kar / Ravikant Pal / Richard Mariadasse / Tanya Parish / Jeyaraman Jeyakanthan / Kutti R Vinothkumar / Bichitra Kumar Biswal /
Abstract: Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The ...Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.
History
DepositionFeb 15, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author / em_3d_fitting_list / pdbx_initial_refinement_model
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_DOI / _citation.pdbx_database_id_PubMed / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Enolase
B: Enolase
C: Enolase
D: Enolase
E: Enolase
F: Enolase
G: Enolase
H: Enolase


Theoretical massNumber of molelcules
Total (without water)367,6998
Polymers367,6998
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20560 Å2
ΔGint-45 kcal/mol
Surface area107030 Å2

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Components

#1: Protein
Enolase / 2-phospho-D-glycerate hydro-lyase / 2-phosphoglycerate dehydratase


Mass: 45962.355 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: eno, AYJ03_005430, DSI38_18100, ERS007663_02163, ERS013471_01432, ERS024276_01771, ERS027659_01730, ERS075361_03197, ERS094182_03347, F6W99_03724, SAMEA2683035_03102
Production host: Mycolicibacterium smegmatis (bacteria) / References: UniProt: A0A0E8NV14, phosphopyruvate hydratase

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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 Enolase enzyme / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.37 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Mycolicibacterium smegmatis (bacteria) / Strain: mc24517
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
2150 mMsodium chlorideNaCl1
325 mMImidazoleImidazole1
41 mMMagnesium sulphateMgSO41
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 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 magnification: 75000 X / Calibrated magnification: 130841 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 27.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 153748
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75126 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 7CLL

7cll


Accession code: 7CLL / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00625334
ELECTRON MICROSCOPYf_angle_d0.57934461
ELECTRON MICROSCOPYf_dihedral_angle_d11.30715095
ELECTRON MICROSCOPYf_chiral_restr0.0443992
ELECTRON MICROSCOPYf_plane_restr0.0044615

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