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

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Basic information

Entry
Database: EMDB / ID: 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.
Sample3,4-dihydroxybenzoate decarboxylase:
Function / homology3-octaprenyl-4-hydroxybenzoate carboxy-lyase / UbiD decarboxylyase family / carboxy-lyase activity / 3,4-dihydroxybenzoate decarboxylase
Function and homology information
SourceEnterobacter cloacae (bacteria)
Methodsingle particle reconstruction / cryo EM / 4.6 Å resolution
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 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 ...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.
DateDeposition: May 12, 2017 / Header (metadata) release: May 31, 2017 / Map release: Sep 13, 2017 / Last update: Oct 25, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • 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

Downloads & links

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Map

Fileemd_3718.map.gz (map file in CCP4 format, 44958 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
224 pix
1.12 Å/pix.
= 250.88 Å
224 pix
1.12 Å/pix.
= 250.88 Å
224 pix
1.12 Å/pix.
= 250.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density
Contour Level:0.02 (by author), 0.02 (movie #1):
Minimum - Maximum-0.037165664 - 0.06892177
Average (Standard dev.)-0.00015808595 (0.0039911764)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions224224224
Origin000
Limit223223223
Spacing224224224
CellA=B=C: 250.88 Å
α=β=γ: 90 deg.

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
start NX/NY/NZ
NX/NY/NZ
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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Sample components

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Entire 3,4-dihydroxybenzoate decarboxylase

EntireName: 3,4-dihydroxybenzoate decarboxylase / Number of components: 2
MassTheoretical: 320 kDa

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Component #1: protein, 3,4-dihydroxybenzoate decarboxylase

ProteinName: 3,4-dihydroxybenzoate decarboxylase / Recombinant expression: No
MassTheoretical: 320 kDa
SourceSpecies: Enterobacter cloacae (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, 3,4-dihydroxybenzoate decarboxylase

ProteinName: 3,4-dihydroxybenzoate decarboxylase / Recombinant expression: No

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.15 mg/ml / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 100 % / Details: 11 second blot

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Electron microscopy imaging

ImagingMicroscope: JEOL 3200FSC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 54 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 30000 X (nominal), 44643 X (calibrated) / Cs: 4.2 mm / Imaging mode: BRIGHT FIELD / Energy filter: JEOL in-column filter with a width of 20eV
Specimen HolderModel: JEOL 3200FSC CRYOHOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionDetails: 40-frame movies in 8 seconds

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D3 (2*3 fold dihedral) / Number of projections: 22650
3D reconstructionSoftware: RELION / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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