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- PDB-9exv: Broad substrate scope C-C oxidation in cyclodipeptides catalysed ... -

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Basic information

Entry
Database: PDB / ID: 9exv
TitleBroad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament
Components
  • AlbB
  • Nitroreductase
KeywordsOXIDOREDUCTASE / Cyclodipeptides oxidase
Function / homologyProtein of unknown function DUF6092 / Family of unknown function (DUF6092) / Nitroreductase / Nitroreductase family / Nitroreductase-like / oxidoreductase activity / FLAVIN MONONUCLEOTIDE / Nitroreductase / AlbB
Function and homology information
Biological speciesNocardiopsis dassonvillei (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3 Å
AuthorsSutherland, E. / Sundaramoorthy, R. / Czekster, C.M.
Funding support United Kingdom, 1items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitroreductase
B: Nitroreductase
C: AlbB
D: AlbB
E: AlbB
F: AlbB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,2218
Polymers88,3086
Non-polymers9132
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Nitroreductase


Mass: 21234.223 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nocardiopsis dassonvillei (bacteria) / Gene: Ndas_1146
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: D7B1W6
#2: Protein
AlbB


Mass: 11459.994 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nocardiopsis dassonvillei (bacteria) / Gene: Ndas_1147
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: D7B1W7
#3: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Filament of Nocardiopsis dassonvillei Cyclodipeptide enzyme
Type: COMPLEX / Entity ID: #2, #1 / Source: RECOMBINANT
Molecular weightValue: 0.33 kDa/nm / Experimental value: NO
Source (natural)Organism: Nocardiopsis dassonvillei (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 9
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMSodium chlorideNacl1
220 mMTris(hydroxymethyl)aminomethaneTris1
35 mMdithioerythritolDTT1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Mono disperse filament made of two subunits A and B of Cyclodipeptide enzyme from Nocardiopsis dassonvillei
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 70 K
Image recordingAverage exposure time: 1.79 sec. / Electron dose: 33 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12900
Image scansWidth: 5000 / Height: 4000

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 132.37 ° / Axial rise/subunit: 45.45 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1180140
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 849600 / Algorithm: FOURIER SPACE / Num. of class averages: 15 / Symmetry type: HELICAL
Atomic model buildingB value: 179 / Protocol: AB INITIO MODEL / Space: REAL / Details: Refinement is done in Phenix
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 77.36 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315808
ELECTRON MICROSCOPYf_angle_d0.52517893
ELECTRON MICROSCOPYf_chiral_restr0.0374892
ELECTRON MICROSCOPYf_plane_restr0.00521047
ELECTRON MICROSCOPYf_dihedral_angle_d13.23072191

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