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- PDB-7clk: Mycobacterium tuberculosis enolase in complex with alternate 2-ph... -

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Basic information

Entry
Database: PDB / ID: 7clk
TitleMycobacterium tuberculosis enolase in complex with alternate 2-phosphoglycerate
ComponentsEnolase
KeywordsLYASE / Mycobacterium tuberculosis / enolase / complex / 2-phosphoglycerate
Function / homology
Function and homology information


phosphopyruvate hydratase / phosphopyruvate hydratase complex / phosphopyruvate hydratase activity / glycolytic process / magnesium ion binding / cell surface / 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
2-PHOSPHOGLYCERIC ACID / ACETATE ION / Enolase
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.15 Å
AuthorsAhmad, M. / Jha, B. / Tiwari, S. / Pal, R.K. / Biswal, B.K.
Funding support India, 1items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India) 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
DepositionJul 21, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 26, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 8, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
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.title / _citation.year
Revision 1.2Nov 29, 2023Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Enolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)46,96116
Polymers45,9621
Non-polymers99915
Water3,171176
1
A: Enolase
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)375,692128
Polymers367,6998
Non-polymers7,993120
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
crystal symmetry operation5_555-x,y,-z1
crystal symmetry operation6_555x,-y,-z1
crystal symmetry operation7_555y,x,-z1
crystal symmetry operation8_555-y,-x,-z1
Buried area36760 Å2
ΔGint-261 kcal/mol
Surface area94620 Å2
MethodPISA
Unit cell
Length a, b, c (Å)139.804, 139.804, 89.645
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number97
Space group name H-MI422
Components on special symmetry positions
IDModelComponents
11A-731-

HOH

21A-738-

HOH

31A-742-

HOH

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Components

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Protein , 1 types, 1 molecules A

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


Mass: 45962.355 Da / Num. of mol.: 1
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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Non-polymers , 5 types, 191 molecules

#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical
ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H3O2
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Mg
#5: Chemical ChemComp-2PG / 2-PHOSPHOGLYCERIC ACID / 2-Phosphoglyceric acid


Mass: 186.057 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7O7P / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 176 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 2.47 Å3/Da / Density % sol: 50.22 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 20% PEG 3350, 0.2M ammonium acetate, Bis-Tris pH 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E+ SUPERBRIGHT / Wavelength: 1.54178 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Mar 15, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54178 Å / Relative weight: 1
ReflectionResolution: 2→35 Å / Num. obs: 29616 / % possible obs: 97.9 % / Redundancy: 4.7 % / Rmerge(I) obs: 0.08 / Net I/σ(I): 13.39
Reflection shellResolution: 2→2.07 Å / Rmerge(I) obs: 0.545 / Num. unique obs: 2875 / % possible all: 96.4

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5j04

5j04


Resolution: 2.15→23.99 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.947 / SU B: 10.214 / SU ML: 0.115 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.188 / ESU R Free: 0.161 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.1908 1143 4.8 %RANDOM
Rwork0.1425 ---
obs0.1448 22819 98.05 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 74.42 Å2 / Biso mean: 27.325 Å2 / Biso min: 15.2 Å2
Baniso -1Baniso -2Baniso -3
1-0.21 Å20 Å20 Å2
2--0.21 Å20 Å2
3----0.42 Å2
Refinement stepCycle: final / Resolution: 2.15→23.99 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3095 0 64 176 3335
Biso mean--50.12 36.13 -
Num. residues----423
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0150.0133225
X-RAY DIFFRACTIONr_bond_other_d0.0010.0173011
X-RAY DIFFRACTIONr_angle_refined_deg1.5031.644379
X-RAY DIFFRACTIONr_angle_other_deg1.3621.5736953
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.2595434
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.72822.739157
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.21315484
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.391521
X-RAY DIFFRACTIONr_chiral_restr0.080.2431
X-RAY DIFFRACTIONr_gen_planes_refined0.0120.023729
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02638
LS refinement shellResolution: 2.15→2.206 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.241 81 -
Rwork0.196 1656 -
all-1737 -
obs--97.58 %
Refinement TLS params.Method: refined / Origin x: 15.3662 Å / Origin y: -41.467 Å / Origin z: 3.6108 Å
111213212223313233
T0.012 Å2-0.014 Å2-0.0074 Å2-0.0215 Å20.0002 Å2--0.0182 Å2
L0.0638 °2-0.018 °20.0329 °2-0.1544 °2-0.0056 °2--0.3182 °2
S0.0127 Å °0.0015 Å °-0.033 Å °0.0012 Å °-0.0045 Å °0.0011 Å °-0.0442 Å °0.0733 Å °-0.0082 Å °

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