+Open data
-Basic information
Entry | Database: PDB / ID: 6jql | |||||||||||||||
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Title | Structure of PaaZ, a bifunctional enzyme | |||||||||||||||
Components | Bifunctional protein PaaZ | |||||||||||||||
Keywords | HYDROLASE / substrate channeling / bi-functional enzyme / dehydrogenase | |||||||||||||||
Function / homology | Function and homology information 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase / oxepin-CoA hydrolase / hydrolase activity, acting on acid carbon-carbon bonds, in ketonic substances / ether hydrolase activity / 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 | |||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | |||||||||||||||
Authors | Gakher, L. / Vinothkumar, K.R. / Katagihallimath, N. / Sowdhamini, R. / Sathyanarayanan, N. / Cannone, G. | |||||||||||||||
Funding support | India, United Kingdom, 4items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6jql.cif.gz | 649.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6jql.ent.gz | 547.5 KB | Display | PDB format |
PDBx/mmJSON format | 6jql.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6jql_validation.pdf.gz | 984.4 KB | Display | wwPDB validaton report |
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Full document | 6jql_full_validation.pdf.gz | 1016.3 KB | Display | |
Data in XML | 6jql_validation.xml.gz | 106.6 KB | Display | |
Data in CIF | 6jql_validation.cif.gz | 165 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/jq/6jql ftp://data.pdbj.org/pub/pdb/validation_reports/jq/6jql | HTTPS FTP |
-Related structure data
Related structure data | 9873MC 9874C 9875C 9876C 6jqmC 6jqnC 6jqoC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: PaaZ / Type: COMPLEX Details: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity. Entity ID: all / Source: RECOMBINANT | ||||||||||||
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Molecular weight | Value: 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 solution | pH: 7.4 Details: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl | ||||||||||||
Buffer component |
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Specimen | Conc.: 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 support | Details: 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 | ||||||||||||
Vitrification | Instrument: 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Data was collected with EPU software |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K |
Image recording | Average 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 scans | Sampling size: 14 µm / Width: 4096 / Height: 4096 |
-Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Details: Particles were automatically corrected by Relion during data processing Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 118237 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | B value: 44.4 / Protocol: OTHER / Space: REAL Details: Real space refinement with secondary structure enabled, minimization and adp |