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- PDB-6jqo: Structure of PaaZ, a bifunctional enzyme in complex with NADP+ an... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6jqo | |||||||||||||||
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Title | Structure of PaaZ, a bifunctional enzyme in complex with NADP+ and CCoA | |||||||||||||||
![]() | Bifunctional protein PaaZ | |||||||||||||||
![]() | HYDROLASE / substrate channeling / bi-functional enzyme / dehydrogenase | |||||||||||||||
Function / homology | ![]() 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 | ![]() ![]() | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||||||||
![]() | Gakher, L. / Vinothkumar, K.R. / Katagihallimath, N. / Sowdhamini, R. / Sathyanarayanan, N. / Cannone, G. | |||||||||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 665.6 KB | Display | ![]() |
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PDB format | ![]() | 563.7 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.6 MB | Display | ![]() |
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Full document | ![]() | 1.7 MB | Display | |
Data in XML | ![]() | 112.6 KB | Display | |
Data in CIF | ![]() | 168 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9876MC ![]() 9873C ![]() 9874C ![]() 9875C ![]() 6jqlC ![]() 6jqmC ![]() 6jqnC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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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.) ![]() ![]() ![]() ![]() References: UniProt: P77455, oxepin-CoA hydrolase, 3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase #2: Chemical | ChemComp-NAP / #3: Chemical | ChemComp-COO / |
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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 |
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Sample preparation
Component | Name: PaaZ / Type: COMPLEX Details: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity. Entity ID: #1 / Source: RECOMBINANT | ||||||||||||
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Molecular weight | Value: 0.44 MDa / Experimental value: NO | ||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||
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. NADP+ and CCoA were added 10 fold excess and incubated for 15 minutes before applying to the grid. | ||||||||||||
Specimen support | Details: Grids were glow discharged for 5 minutes and graphene oxide was applied. This was followed by 3 times wash with water and dried. 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 time 1. |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 134615 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: 563 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 |
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Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Counting mode | ||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTF was corrected per particle within relion during processing Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 122910 | ||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120968 / 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 |