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- PDB-6jql: Structure of PaaZ, a bifunctional enzyme -

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Basic information

Entry
Database: PDB / ID: 6jql
TitleStructure of PaaZ, a bifunctional enzyme
ComponentsBifunctional protein PaaZ
KeywordsHYDROLASE / substrate channeling / bi-functional enzyme / dehydrogenase
Function / homology
Function and homology information


3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase / oxepin-CoA hydrolase / ether hydrolase activity / hydrolase activity, acting on acid carbon-carbon bonds, in ketonic substances / oxidoreductase activity, acting on CH or CH2 groups, NAD or NADP as acceptor / phenylacetate catabolic process / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / enoyl-CoA hydratase activity / identical protein binding
Similarity search - Function
Phenylacetic acid degradation protein PaaN / MaoC-like dehydratase domain / MaoC like domain / Hotdog Thioesterase / Thiol Ester Dehydrase; Chain A / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde Dehydrogenase; Chain A, domain 1 / HotDog domain superfamily ...Phenylacetic acid degradation protein PaaN / MaoC-like dehydratase domain / MaoC like domain / Hotdog Thioesterase / Thiol Ester Dehydrase; Chain A / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 2 / Aldehyde Dehydrogenase; Chain A, domain 1 / Aldehyde Dehydrogenase; Chain A, domain 1 / HotDog domain superfamily / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, C-terminal / Aldehyde dehydrogenase, N-terminal / Aldehyde/histidinol dehydrogenase / Roll / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Bifunctional protein PaaZ
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGakher, L. / Vinothkumar, K.R. / Katagihallimath, N. / Sowdhamini, R. / Sathyanarayanan, N. / Cannone, G.
Funding support India, United Kingdom, 4items
OrganizationGrant numberCountry
Department of Biotechnology (India)DBT/PR12422/MED/31/287/2014 India
Council of Scientific & Industrial ResearchCSIR/37/1606/13/EMR-II India
Department of Biotechnology (India)BT/PR5081/INF/22/156/2012 India
Medical Research Council (United Kingdom)U105184322 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.
Authors: Nitish Sathyanarayanan / Giuseppe Cannone / Lokesh Gakhar / Nainesh Katagihallimath / Ramanathan Sowdhamini / Subramanian Ramaswamy / Kutti R Vinothkumar /
Abstract: Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been ...Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.
History
DepositionMar 31, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / Item: _citation.title
Revision 1.2Sep 25, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Bifunctional protein PaaZ
B: Bifunctional protein PaaZ
C: Bifunctional protein PaaZ
D: Bifunctional protein PaaZ
E: Bifunctional protein PaaZ
F: Bifunctional protein PaaZ


Theoretical massNumber of molelcules
Total (without water)443,8166
Polymers443,8166
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, Oligomeric state with gel filtration, Dynamic light scattering or Small-angle X-ray scattering indicated it is an oligomer but did not yield unambiguous results., light scattering, ...Evidence: SAXS, Oligomeric state with gel filtration, Dynamic light scattering or Small-angle X-ray scattering indicated it is an oligomer but did not yield unambiguous results., light scattering, Oligomeric state with gel filtration, Dynamic light scattering or Small-angle X-ray scattering indicated it is an oligomer but did not yield unambiguous results.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37590 Å2
ΔGint-159 kcal/mol
Surface area133520 Å2

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Components

#1: Protein
Bifunctional protein PaaZ


Mass: 73969.391 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: paaZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P77455, oxepin-CoA hydrolase

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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: PaaZ / Type: COMPLEX
Details: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.44 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) / Plasmid: PET 28a
Buffer solutionpH: 7.4
Details: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
250 mMNACL1
SpecimenConc.: 0.015 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The peak fraction from gel filtration was used for grid preparation.
Specimen supportDetails: The Ultrafoil grids was glow discharged in air for 5 minutes. Subsequently, 0.2 mg/ml of graphene oxide solution was placed and washed twice with water. The grids were air dried and used directly for freezing.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K
Details: blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting times 1.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Data was collected with EPU software
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 132075 X / Nominal defocus max: 3200 nm / Nominal defocus min: 2200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 60 sec. / Electron dose: 27 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 552
Details: The 60 second exposure was saved into 75 frames with each frame ~0.36 e-. The frames were then grouped into 3 for alignment and summed images were used for data processing
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatch0.56particle selection
2EPU1.7image acquisitionOne image per hole was collected
4Gctf1.06CTF correction
7Coot0.8model fitting
9PHENIX1.13model refinement
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
CTF correctionDetails: Particles were automatically corrected by Relion during data processing
Type: NONE
Particle selectionNum. of particles selected: 118237
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86420 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 44.4 / Protocol: OTHER / Space: REAL
Details: Real space refinement with secondary structure enabled, minimization and adp

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