[English] 日本語
Yorodumi
- EMDB-9875: Structure of PaaZ, a bifunctional enzyme in complex with NADP+ an... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-9875
TitleStructure of PaaZ, a bifunctional enzyme in complex with NADP+ and OCoA
Map dataThis map is of PaaZ in complex with NADP+ and Octanoyl CoA used for interpretation.
Sample
  • Complex: PaaZ
    • Protein or peptide: Bifunctional protein PaaZ
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: OCTANOYL-COENZYME A
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 / enoyl-CoA hydratase activity / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / 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, N-terminal / Aldehyde dehydrogenase, C-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: 3.1 Å
AuthorsGakher L / Vinothkumar KR / Katagihallimath N / Sowdhamini R / Sathyanarayanan N / Cannone G
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

-
Structure visualization

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

emd_9875.png

Downloads & links

-
Map

FileDownload / File: emd_9875.map.gz / Format: CCP4 / Size: 266.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map is of PaaZ in complex with NADP+ and Octanoyl CoA used for interpretation.
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.085 / Movie #1: 0.1
Minimum - Maximum-0.3496194 - 0.79728407
Average (Standard dev.)0.0001101211 (±0.016257212)
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-0.3500.7970.000

-
Supplemental data

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

Fileemd_9875_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)

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

Fileemd_9875_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)

-
Sample components

-
Entire : PaaZ

EntireName: PaaZ
Components
  • Complex: PaaZ
    • Protein or peptide: Bifunctional protein PaaZ
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: OCTANOYL-COENZYME A

-
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

-
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

-
Macromolecule #2: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 2 / Number of copies: 6 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

-
Macromolecule #3: OCTANOYL-COENZYME A

MacromoleculeName: OCTANOYL-COENZYME A / type: ligand / ID: 3 / Number of copies: 6 / Formula: CO8
Molecular weightTheoretical: 893.73 Da
Chemical component information

ChemComp-CO8:
OCTANOYL-COENZYME A / Octanoyl-CoA

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

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

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 were glow discharged for 5 minutes and then graphene oxide was applied. Subsequently, grids were washed with water 3 times and dried. The graphene oxide grids were then used for freezing.
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. NADP+ and OCoA were added 10 fold excess and incubated for 15 minutes before freezing.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
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 FIELDBright-field microscopy / 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
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: 667 / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 179346
Startup modelType of model: OTHER
Details: The map from native enzyme (D_13000114900) was low pass filtered to 60A and used as reference.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 101503
Detailscounting mode was used
FSC plot (resolution estimation)

-
Atomic model buiding 1

DetailsReal space refinement with secondary structure enabled, minimization and adp
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 59.9
Output model

PDB-6jqn:
Structure of PaaZ, a bifunctional enzyme in complex with NADP+ and OCoA

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more