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- EMDB-3718: 3,4-dihydroxybenzoate decarboxylase AroY from Enterobacter cloaca... -

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Basic information

Entry
Database: EMDB / ID: EMD-3718
Title3,4-dihydroxybenzoate decarboxylase AroY from Enterobacter cloacae in the apo state
Map dataSingle-particle cryo-EM map of the 3,4-dihydroxybenzoate decarboxylase AroY from Enterobacter cloacae in the apo state.
Sample
  • Complex: 3,4-dihydroxybenzoate decarboxylase
    • Protein or peptide: 3,4-dihydroxybenzoate decarboxylase
Biological speciesEnterobacter cloacae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsBaerland N / Kaltwasser S / Vonck J / Pavkov-Keller T
CitationJournal: Angew Chem Int Ed Engl / Year: 2017
Title: Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase.
Authors: Stefan E Payer / Stephen A Marshall / Natalie Bärland / Xiang Sheng / Tamara Reiter / Andela Dordic / Georg Steinkellner / Christiane Wuensch / Susann Kaltwasser / Karl Fisher / Stephen E J ...Authors: Stefan E Payer / Stephen A Marshall / Natalie Bärland / Xiang Sheng / Tamara Reiter / Andela Dordic / Georg Steinkellner / Christiane Wuensch / Susann Kaltwasser / Karl Fisher / Stephen E J Rigby / Peter Macheroux / Janet Vonck / Karl Gruber / Kurt Faber / Fahmi Himo / David Leys / Tea Pavkov-Keller / Silvia M Glueck /
Abstract: The utilization of CO as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para- ...The utilization of CO as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.
History
DepositionMay 12, 2017-
Header (metadata) releaseMay 31, 2017-
Map releaseSep 13, 2017-
UpdateOct 25, 2017-
Current statusOct 25, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3718.png

Downloads & links

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Map

FileDownload / File: emd_3718.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM map of the 3,4-dihydroxybenzoate decarboxylase AroY from Enterobacter cloacae in the apo state.
Voxel sizeX=Y=Z: 1.12 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.037165664 - 0.06892177
Average (Standard dev.)-0.00015808595 (±0.0039911764)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 250.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.121.121.12
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z250.880250.880250.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0370.069-0.000

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Supplemental data

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Half map: half-map2 of 3,4-dihydroxybenzoate decarboxylase AroY

Fileemd_3718_half_map_1.map
Annotationhalf-map2 of 3,4-dihydroxybenzoate decarboxylase AroY
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map1 of 3,4-dihydroxybenzoate decarboxylase AroY

Fileemd_3718_half_map_2.map
Annotationhalf-map1 of 3,4-dihydroxybenzoate decarboxylase AroY
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 3,4-dihydroxybenzoate decarboxylase

EntireName: 3,4-dihydroxybenzoate decarboxylase
Components
  • Complex: 3,4-dihydroxybenzoate decarboxylase
    • Protein or peptide: 3,4-dihydroxybenzoate decarboxylase

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Supramolecule #1: 3,4-dihydroxybenzoate decarboxylase

SupramoleculeName: 3,4-dihydroxybenzoate decarboxylase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Enterobacter cloacae (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 320 KDa

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Macromolecule #1: 3,4-dihydroxybenzoate decarboxylase

MacromoleculeName: 3,4-dihydroxybenzoate decarboxylase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MQNPINDLRS AIALLQRHPG HYIETDHPVD PNAELAGVYR HIGAGGTVKR PTRTGPAMMF NSVKGYPGS RILVGMHASR ERAALLLGCV PSKLAQHVGQ AVKNPVAPVV VPASQAPCQE Q VFYADDPD FDLRKLLPAP TNTPIDAGPF FCLGLVLASD PEDTSLTDVT ...String:
MQNPINDLRS AIALLQRHPG HYIETDHPVD PNAELAGVYR HIGAGGTVKR PTRTGPAMMF NSVKGYPGS RILVGMHASR ERAALLLGCV PSKLAQHVGQ AVKNPVAPVV VPASQAPCQE Q VFYADDPD FDLRKLLPAP TNTPIDAGPF FCLGLVLASD PEDTSLTDVT IHRLCVQERD EL SMFLAAG RHIEVFRKKA EAAGKPLPVT INMGLDPAIY IGACFEAPTT PFGYNELGVA GAL RQQPVE LVQGVAVKEK AIARAEIIIE GELLPGVRVR EDQHTNTGHA MPEFPGYCGE ANPS LPVIK VKAVTMRNHA ILQTLVGPGE EHTTLAGLPT EASIRNAVEE AIPGFLQNVY AHTAG GGKF LGILQVKKRQ PSDEGRQGQA ALIALATYSE LKNIILVDED VDIFDSDDIL WAMTTR MQG DVSITTLPGI RGHQLDPSQS PDYSTSIRGN GISCKTIFDC TVPWALKARF ERAPFME VD PTPWAPELFS DKK

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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

Concentration0.15 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: 11 second blot.

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 44643 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.2 mm / Nominal magnification: 30000
Specialist opticsEnergy filter - Name: JEOL in-column filter with a width of 20eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 8.0 sec. / Average electron dose: 54.0 e/Å2 / Details: 40-frame movies in 8 seconds

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Image processing

CTF correctionSoftware - Name: Gctf / Software - details: per-particle ctf
Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 22650
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe crystal structure of the same molecule was rigid-body fitted in the map.
RefinementProtocol: RIGID BODY FIT

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