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- EMDB-9874: Structure of PaaZ with NADPH -

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Basic information

Entry
Database: EMDB / ID: EMD-9874
TitleStructure of PaaZ with NADPH
Map dataThis map is of PaaZ with NADPH bound.
Sample
  • Complex: PaaZ
    • Protein or peptide: Bifunctional protein PaaZ
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
Keywordssubstrate channeling / bi-functional enzyme / hydrolase / 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
Phenylacetic acid degradation protein PaaN / MaoC-like dehydratase domain / MaoC like domain / HotDog domain superfamily / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, C-terminal / Aldehyde dehydrogenase, N-terminal / Aldehyde/histidinol dehydrogenase
Similarity search - Domain/homology
Bifunctional protein PaaZ
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGakher L / Cannone G / Katagihallimath N / Sowdhamini R / Sathyanarayanan N
Funding support India, United Kingdom, 4 items
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, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6jqm
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9874.png

Downloads & links

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Map

FileDownload / File: emd_9874.map.gz / Format: CCP4 / Size: 266.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map is of PaaZ with NADPH bound.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 412 pix.
= 436.72 Å		1.06 Å/pix.
x 412 pix.
= 436.72 Å		1.06 Å/pix.
x 412 pix.
= 436.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.1
Minimum - Maximum-0.39878255 - 0.6848086
Average (Standard dev.)0.000091537215 (±0.014926524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions412412412
Spacing412412412
CellA=B=C: 436.71997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z412412412
origin x/y/z0.0000.0000.000
length x/y/z436.720436.720436.720
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS412412412
D min/max/mean-0.3990.6850.000

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Supplemental data

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Additional map: This map is one of the half-maps after refinement.

Fileemd_9874_additional_1.map
AnnotationThis map is one of the half-maps after refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: This map is one of the half-maps after refinement.

Fileemd_9874_additional_2.map
AnnotationThis map is one of the half-maps after refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PaaZ

EntireName: PaaZ
Components
  • Complex: PaaZ
    • Protein or peptide: Bifunctional protein PaaZ
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Supramolecule #1: PaaZ

SupramoleculeName: PaaZ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: PaaZ is a bifunctional enzyme that has hydrolase and dehydrogenase activity.
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 440 KDa

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Macromolecule #1: Bifunctional protein PaaZ

MacromoleculeName: Bifunctional protein PaaZ / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: oxepin-CoA hydrolase
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Molecular weightTheoretical: 73.969391 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHQQ LASFLSGTWQ SGRGRSRLIH HAISGEALWE VTSEGLDMAA ARQFAIEKGA PALRAMTFIE RAAMLKAVAK HLLSEKERF YALSAQTGAT RADSWVDIEG GIGTLFTYAS LGSRELPDDT LWPEDELIPL SKEGGFAARH LLTSKSGVAV H INAFNFPC ...String:
MGHHHHHHQQ LASFLSGTWQ SGRGRSRLIH HAISGEALWE VTSEGLDMAA ARQFAIEKGA PALRAMTFIE RAAMLKAVAK HLLSEKERF YALSAQTGAT RADSWVDIEG GIGTLFTYAS LGSRELPDDT LWPEDELIPL SKEGGFAARH LLTSKSGVAV H INAFNFPC WGMLEKLAPT WLGGMPAIIK PATATAQLTQ AMVKSIVDSG LVPEGAISLI CGSAGDLLDH LDSQDVVTFT GS AATGQML RVQPNIVAKS IPFTMEADSL NCCVLGEDVT PDQPEFALFI REVVREMTTK AGQKCTAIRR IIVPQALVNA VSD ALVARL QKVVVGDPAQ EGVKMGALVN AEQRADVQEK VNILLAAGCE IRLGGQADLS AAGAFFPPTL LYCPQPDETP AVHA TEAFG PVATLMPAQN QRHALQLACA GGGSLAGTLV TADPQIARQF IADAARTHGR IQILNEESAK ESTGHGSPLP QLVHG GPGR AGGGEELGGL RAVKHYMQRT AVQGSPTMLA AISKQWVRGA KVEEDRIHPF RKYFEELQPG DSLLTPRRTM TEADIV NFA CLSGDHFYAH MDKIAAAESI FGERVVHGYF VLSAAAGLFV DAGVGPVIAN YGLESLRFIE PVKPGDTIQV RLTCKRK TL KKQRSAEEKP TGVVEWAVEV FNQHQTPVAL YSILTLVARQ HGDFVD

UniProtKB: Bifunctional protein PaaZ

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Macromolecule #2: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
type: ligand / ID: 2 / Number of copies: 6 / Formula: NDP
Molecular weightTheoretical: 745.421 Da
Chemical component information

ChemComp-NDP:
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 7.4 / Component:
ConcentrationName
25.0 mMHEPES
50.0 mMNACL

Details: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa
Details: Grids was glow discharged for 5 minutes and then graphene oxide was applied. The grids were washed with water 3 times and dried before using.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
Details: blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting times 1..
DetailsThe peak fraction from gel filtration was used for grid preparation. 10-fold excess of NADPH was added before grid freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
DetailsData was collected with EPU software
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 732 / Average exposure time: 60.0 sec. / Average electron dose: 27.0 e/Å2
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 132075 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 2.2 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailscounting mode
Particle selectionNumber selected: 175482
Startup modelType of model: OTHER
Details: PaaZ native map lowpass filtered to 60A was used as reference
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 103298
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsReal space refinement with secondary structure enabled, minimization and adp
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 68.9
Output model

PDB-6jqm:
Structure of PaaZ with NADPH

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