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- EMDB-42375: Deinococcus aerius TR0125 C-glucosyl deglycosidase (CGD), cryo-EM -

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Basic information

Entry
Database: EMDB / ID: EMD-42375
TitleDeinococcus aerius TR0125 C-glucosyl deglycosidase (CGD), cryo-EM
Map dataRefinement map.
Sample
  • Complex: C-glucosyl deglycosidase, alpha subunit
    • Protein or peptide: Xylose isomerase-like TIM barrel domain-containing protein
    • Protein or peptide: DUF6379 domain-containing protein
  • Ligand: MANGANESE (II) ION
Keywordsbacterial C-glucosyl deglycosidase / C-C bond cleavage / C-glucosyl aromatic polyketides / C-glucosyl flavonoids / Deinococcus aerius / soil bacterium / N-terminal DUF6379 beta-sandwich domain / C-terminal TIM-barrel domain / alpha2beta2 heterotetramer / LYASE
Function / homologyDomain of unknown function DUF6379 / C-glycoside deglycosidase beta subunit / : / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily / lyase activity / Xylose isomerase-like TIM barrel domain-containing protein / C-deglycosylation enzyme beta subunit
Function and homology information
Biological speciesDeinococcus aerius (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsFurlanetto V / Kalyani DC / Kostelac A / Haltrich D / Hallberg BM / Divne C
Funding support Sweden, Austria, 5 items
OrganizationGrant numberCountry
Other governmentFormas 2017-00983 Sweden
Other governmentVR 2017-03877 Sweden
Other privateOscar and Lili Lamm Memorial Foundation DO2017-0020 Sweden
Austrian Science FundFWF P33545 Austria
Austrian Science FundFWF W1224 Austria
CitationJournal: J Mol Biol / Year: 2024
Title: Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius.
Authors: Valentina Furlanetto / Dayanand C Kalyani / Anja Kostelac / Jolanta Puc / Dietmar Haltrich / B Martin Hällberg / Christina Divne /
Abstract: Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or ...Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C-C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an αβ heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C-C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, K 0.047 mM, k 51 min; DaCGO1/DaCGD, K 0.083 mM, k 0.42 min).
History
DepositionOct 17, 2023-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42375.png

Downloads & links

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Map

FileDownload / File: emd_42375.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefinement map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 258.56 Å		1.01 Å/pix.
x 256 pix.
= 258.56 Å		1.01 Å/pix.
x 256 pix.
= 258.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.049786236 - 1.75417
Average (Standard dev.)0.0008591428 (±0.021085763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42375_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap B

Fileemd_42375_half_map_1.map
Annotationhalfmap B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap A

Fileemd_42375_half_map_2.map
Annotationhalfmap A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : C-glucosyl deglycosidase, alpha subunit

EntireName: C-glucosyl deglycosidase, alpha subunit
Components
  • Complex: C-glucosyl deglycosidase, alpha subunit
    • Protein or peptide: Xylose isomerase-like TIM barrel domain-containing protein
    • Protein or peptide: DUF6379 domain-containing protein
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: C-glucosyl deglycosidase, alpha subunit

SupramoleculeName: C-glucosyl deglycosidase, alpha subunit / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The complex contains two alpha subunits and two beta subunits that form an alpha2beta2 heteroteramer
Source (natural)Organism: Deinococcus aerius (bacteria) / Strain: TR0125

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Macromolecule #1: Xylose isomerase-like TIM barrel domain-containing protein

MacromoleculeName: Xylose isomerase-like TIM barrel domain-containing protein
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Deinococcus aerius (bacteria) / Strain: TR0125
Molecular weightTheoretical: 40.554328 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString: MTQPQIKRGV SLYSFQEEFF LRKMTLEDCV AACASMGAYG IESLAEQMMP GFPNLDDAFY DGWHAMMAKY GTVSVCHDMF LDTKKFRGR LMTLDEQVES FVRDIRHASR LGCTVIRVLN FVSPELMEKV LPHAEQSNMR LGLEIHAPMH FEHPWVLRHI E FMDRLGSP ...String:
MTQPQIKRGV SLYSFQEEFF LRKMTLEDCV AACASMGAYG IESLAEQMMP GFPNLDDAFY DGWHAMMAKY GTVSVCHDMF LDTKKFRGR LMTLDEQVES FVRDIRHASR LGCTVIRVLN FVSPELMEKV LPHAEQSNMR LGLEIHAPMH FEHPWVLRHI E FMDRLGSP LLGFIPDMGI FTKHFPPVMA ERLIRQGATP HIIEYIREQY DRRVLAEYVV GDVRNMGGNP VDIRAAEMLR HN NWSNPRR LLEHMDRIFH VHAKFYEMDE QDRETSLGYE EVIPVLKEGG YSGYLASEYE GNRHIQDAFE VDSVEQVRRH QRM LARLIG EREVAHVAEN LYFQSHHHHH H

UniProtKB: Xylose isomerase-like TIM barrel domain-containing protein

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Macromolecule #2: DUF6379 domain-containing protein

MacromoleculeName: DUF6379 domain-containing protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Deinococcus aerius (bacteria) / Strain: TR0125
Molecular weightTheoretical: 13.755647 KDa
Recombinant expressionOrganism: Escherichia coli B (bacteria)
SequenceString:
MFDKYIVVED SLKRVPGGVQ FGVRLPYYRG LGLSMVETMD VTVDGERVPE ENLTVTLGDR TVPFARRDDE TDTIWNFGEI ATVTARLPH ELGPGEHQVG VNFGLRISYF PVPMVGQDAK TLKL

UniProtKB: C-deglycosylation enzyme beta subunit

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Macromolecule #3: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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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

Concentration1 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
DetailsData collected at the Karolinska Institutet 3D-EM facility
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2153 / Average exposure time: 1.5 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.2 µm
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

Particle selectionNumber selected: 335185
Startup modelType of model: NONE / Details: Ab-initio
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 101068
Initial angle assignmentType: OTHER
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 4.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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