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- PDB-8urc: Bacillus niacini flavin monooxygenase -

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Basic information

Entry
Database: PDB / ID: 8urc
TitleBacillus niacini flavin monooxygenase
ComponentsFlavin monooxygenase
KeywordsCYTOSOLIC PROTEIN / Monooxygenase / flavin binding
Function / homologyChem-DR9 / FLAVIN-ADENINE DINUCLEOTIDE
Function and homology information
Biological speciesNeobacillus niacini (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsRichardson, B.C. / French, J.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM124898 United States
CitationJournal: Biochemistry / Year: 2024
Title: Structural and Functional Characterization of a Novel Class A Flavin Monooxygenase from .
Authors: Brian C Richardson / Zachary R Turlington / Sofia Vaz Ferreira de Macedo / Sara K Phillips / Kay Perry / Savannah G Brancato / Emmalee W Cooke / Jonathan R Gwilt / Morgan A Dasovich / Andrew ...Authors: Brian C Richardson / Zachary R Turlington / Sofia Vaz Ferreira de Macedo / Sara K Phillips / Kay Perry / Savannah G Brancato / Emmalee W Cooke / Jonathan R Gwilt / Morgan A Dasovich / Andrew J Roering / Francis M Rossi / Mark J Snider / Jarrod B French / Katherine A Hicks /
Abstract: A gene cluster responsible for the degradation of nicotinic acid (NA) in has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to ...A gene cluster responsible for the degradation of nicotinic acid (NA) in has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to establish pathway intermediates. One of the genes within this cluster encodes a flavin monooxygenase (BnFMO) that is hypothesized to catalyze a hydroxylation reaction. Kinetic analyses of the recombinantly purified BnFMO suggest that this enzyme catalyzes the hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine (2,6-DHP), which is formed spontaneously by the decarboxylation of 2,6-DHNA. To understand the details of this hydroxylation reaction, we determined the structure of BnFMO using a multimodel approach combining protein X-ray crystallography and cryo-electron microscopy (cryo-EM). A liganded BnFMO cryo-EM structure was obtained in the presence of 2,6-DHP, allowing us to make predictions about potential catalytic residues. The structural data demonstrate that BnFMO is trimeric, which is unusual for Class A flavin monooxygenases. In both the electron density and coulomb potential maps, a region at the trimeric interface was observed that was consistent with and modeled as lipid molecules. High-resolution mass spectral analysis suggests that there is a mixture of phosphatidylethanolamine and phosphatidylglycerol lipids present. Together, these data provide insights into the molecular details of the central hydroxylation reaction unique to the aerobic degradation of NA in .
History
DepositionOct 25, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 18, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin ...citation / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Flavin monooxygenase
B: Flavin monooxygenase
C: Flavin monooxygenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)162,2009
Polymers157,6023
Non-polymers4,5986
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, assay for oligomerization, Analytical ultracentrifugation indicates an appropriate molecular weight for the predicted trimer
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Flavin monooxygenase


Mass: 52534.094 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neobacillus niacini (bacteria) / Gene: FMO / Production host: Escherichia coli (E. coli) / Strain (production host): B834(DE3)
#2: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#3: Chemical ChemComp-DR9 / 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL / (2R)-3-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-2-[(9E)-HEXADEC-9-ENOYLOXY]PROPYL (9E)-OCTADEC-9-ENOATE


Mass: 746.991 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C40H75O10P / 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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Flavin monooxygenase trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.165 MDa / Experimental value: YES
Source (natural)Organism: Neobacillus niacini (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: B834(DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
250 mMsodium chlorideNaCl1
31 mMdithiothreitol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4176
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2EPU3image acquisition
4cryoSPARC3.3.1CTF correction
7PHENIX1.21model fitting
9PHENIX1.21model refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3395411 / Details: Template based picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 432941 / Algorithm: FOURIER SPACE / Num. of class averages: 26 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE

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