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- EMDB-50049: Broad substrate scope C-C oxidation in cyclodipeptides catalysed ... -

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Basic information

Entry
Database: EMDB / ID: EMD-50049
TitleBroad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament
Map dataReconstructed reconstructed map
Sample
  • Complex: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme
    • Protein or peptide: AlbB
    • Protein or peptide: Nitroreductase
  • Ligand: FLAVIN MONONUCLEOTIDE
KeywordsCyclodipeptides oxidase / OXIDOREDUCTASE
Function / homologyProtein of unknown function DUF6092 / Family of unknown function (DUF6092) / Nitroreductase / Nitroreductase family / Nitroreductase-like / oxidoreductase activity / Nitroreductase / AlbB
Function and homology information
Biological speciesNocardiopsis dassonvillei (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsSutherland E / Sundaramoorthy R / Czekster CM
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament.
Authors: Emmajay Sutherland / Christopher J Harding / Tancrède du Monceau de Bergendal / Gordon J Florence / Katrin Ackermann / Bela E Bode / Silvia Synowsky / Ramasubramanian Sundaramoorthy / Clarissa Melo Czekster /
Abstract: Cyclic dipeptides are produced by organisms across all domains of life, with many exhibiting anticancer and antimicrobial properties. Oxidations are often key to their biological activities, ...Cyclic dipeptides are produced by organisms across all domains of life, with many exhibiting anticancer and antimicrobial properties. Oxidations are often key to their biological activities, particularly C-C bond oxidation catalysed by tailoring enzymes including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their intricate three-dimensional arrangement involving multiple copies of two distinct small subunits, and mechanistic details underlying substrate selection and catalysis are lacking. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), a component of the biosynthetic pathway for nocazine natural products. We demonstrated that NdasCDO forms filaments in solution, with a covalently bound flavin mononucleotide (FMN) cofactor at the interface between three distinct subunits. The enzyme exhibits promiscuity, processing various cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH and involves the formation of a radical intermediate. Pre-steady-state kinetics, a significant solvent kinetic isotope effect, and the absence of viscosity effects suggested that a step linked to FMN regeneration controlled the reaction rate. Our work elucidates the complex mechanistic and structural characteristics of this dehydrogenation reaction, positioning NdasCDO as a promising biocatalyst and expanding the FMN-dependent oxidase family to include enzyme filaments.
History
DepositionApr 8, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50049.png

Downloads & links

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Map

FileDownload / File: emd_50049.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstructed reconstructed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 340 pix.
= 282.54 Å		0.83 Å/pix.
x 340 pix.
= 282.54 Å		0.83 Å/pix.
x 340 pix.
= 282.54 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.831 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-3.829792 - 5.634334
Average (Standard dev.)0.0016402348 (±0.090768665)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 282.53998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50049_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: Reconstructed map

Fileemd_50049_additional_1.map
AnnotationReconstructed map
Projections & Slices
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Histogram

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Additional map: Focused Map

Fileemd_50049_additional_2.map
AnnotationFocused Map
Projections & Slices
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Additional map: Focused map

Fileemd_50049_additional_3.map
AnnotationFocused map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Maps

Fileemd_50049_half_map_1.map
AnnotationHalf Maps
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half Maps

Fileemd_50049_half_map_2.map
AnnotationHalf Maps
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AxesZYX

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Sample components

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Entire : Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme

EntireName: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme
Components
  • Complex: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme
    • Protein or peptide: AlbB
    • Protein or peptide: Nitroreductase
  • Ligand: FLAVIN MONONUCLEOTIDE

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Supramolecule #1: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme

SupramoleculeName: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1
Source (natural)Organism: Nocardiopsis dassonvillei (bacteria)
Molecular weightTheoretical: 0.33 kDa/nm

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Macromolecule #1: Nitroreductase

MacromoleculeName: Nitroreductase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Nocardiopsis dassonvillei (bacteria)
Molecular weightTheoretical: 21.234223 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MDTGSSEPDA NRCPSQRSSH ALQTLTTRRA VRAFADRPVD DSLLDPMLDA MLAAPSASNK QAWAFVAVRE RRALRLLRAF SPGIIELPP LVVAACFDRS RAVGGSGNST DSGDSWDEGM LCVAMAVENL LLAAHCLGLG GCPSGSFRRG PVRRLLGLPD H LEPLLLVP ...String:
MDTGSSEPDA NRCPSQRSSH ALQTLTTRRA VRAFADRPVD DSLLDPMLDA MLAAPSASNK QAWAFVAVRE RRALRLLRAF SPGIIELPP LVVAACFDRS RAVGGSGNST DSGDSWDEGM LCVAMAVENL LLAAHCLGLG GCPSGSFRRG PVRRLLGLPD H LEPLLLVP IGHPARPLAP APRRDRNEVV SHERWGTGSS

UniProtKB: Nitroreductase

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Macromolecule #2: AlbB

MacromoleculeName: AlbB / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Nocardiopsis dassonvillei (bacteria)
Molecular weightTheoretical: 11.459994 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MSAGEPEVRQ VGEELLLLAA YLLSSGRGLL DEPRQYGTFR CLDAARRVLA LAAGTGPHHP ELDALRGRMD DVMCGPMGDH ELDTLLDQM CERLATVLED PDVISD

UniProtKB: AlbB

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Macromolecule #3: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 2 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 9
Component:
ConcentrationFormulaName
200.0 mMNaclSodium chloride
20.0 mMTrisTris(hydroxymethyl)aminomethane
5.0 mMDTTdithioerythritol
GridModel: Quantifoil R2/1 / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.0001 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsMono disperse filament made of two subunits A and B of Cyclodipeptide enzyme from Nocardiopsis dassonvillei

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 70.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 12900 / Average exposure time: 1.79 sec. / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.2 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
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

Final reconstructionNumber classes used: 15
Applied symmetry - Helical parameters - Δz: 45.45 Å
Applied symmetry - Helical parameters - Δ&Phi: 132.37 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 849600
Segment selectionNumber selected: 1180140
Startup modelType of model: OTHER
Details: Ab-initio model calculated using SGD embedded in cryosparc
Final angle assignmentType: NOT APPLICABLE / Details: helical
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsRefinement is done in Phenix
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 179
Output model

PDB-9exv:
Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

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