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- PDB-8umc: Deinococcus aerius TR0125 C-glucosyl deglycosidase (CGD), cryo-EM -

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Basic information

Entry
Database: PDB / ID: 8umc
TitleDeinococcus aerius TR0125 C-glucosyl deglycosidase (CGD), cryo-EM
Components
  • DUF6379 domain-containing protein
  • Xylose isomerase-like TIM barrel domain-containing protein
KeywordsLYASE / bacterial 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
Function / homologyDomain of unknown function DUF6379 / Domain of unknown function (DUF6379) / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily / : / Xylose isomerase-like TIM barrel domain-containing protein / DUF6379 domain-containing protein
Function and homology information
Biological speciesDeinococcus aerius (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsFurlanetto, V. / Kalyani, D.C. / Kostelac, A. / Haltrich, D. / Hallberg, B.M. / Divne, C.
Funding support Sweden, Austria, 5items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 29, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Xylose isomerase-like TIM barrel domain-containing protein
B: DUF6379 domain-containing protein
C: Xylose isomerase-like TIM barrel domain-containing protein
D: DUF6379 domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,7306
Polymers108,6204
Non-polymers1102
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, X-ray crystallography, cryo-EM
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 40554.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus aerius (bacteria) / Strain: TR0125 / Gene: DAERI_200053 / Plasmid: pNIC-CTHO / Production host: Escherichia coli B (bacteria) / Strain (production host): (DE3)-T1 / References: UniProt: A0A2I9DAN1
#2: Protein DUF6379 domain-containing protein


Mass: 13755.647 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Deinococcus aerius (bacteria) / Strain: TR0125 / Gene: DAERI_200052 / Plasmid: 2A-T / Production host: Escherichia coli B (bacteria) / Strain (production host): (DE3)-T1 / References: UniProt: A0A2I9E2I0
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: C-glucosyl deglycosidase, alpha subunit / Type: COMPLEX
Details: The complex contains two alpha subunits and two beta subunits that form an alpha2beta2 heteroteramer
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Deinococcus aerius (bacteria) / Strain: TR0125
Source (recombinant)Organism: Escherichia coli B (bacteria) / Strain: (DE3)-T1 / Plasmid: pNIC-CTHO (alpha subunit), 2A-T (beta subunit)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Data collected at the Karolinska Institutet 3D-EM facility
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 200 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2153
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Warp0.9particle selection
2EPU3.2image acquisition
4cryoSPARC4.3.1CTF correction
11cryoSPARC4.3.1final Euler assignment
12cryoSPARC4.3.1classification
13cryoSPARC4.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 335185
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101068 / Algorithm: BACK PROJECTION / Symmetry type: POINT

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