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- EMDB-42490: Bacillus niacini flavin monooxygenase with bound (2,6)DHP -

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Basic information

Entry
Database: EMDB / ID: EMD-42490
TitleBacillus niacini flavin monooxygenase with bound (2,6)DHP
Map dataPrimary map filtered by estimated local resolution
Sample
  • Complex: flavin monooxygenase asymmetric trimer
    • Protein or peptide: Flavin monooxygenase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: pyridine-2,6-diol
  • Ligand: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL
KeywordsMonooxygenase / flavin binding / CYTOSOLIC PROTEIN
Biological speciesNeobacillus niacini (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsRichardson BC / French JB
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseSep 18, 2024-
Map releaseSep 18, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42490.png

Downloads & links

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Map

FileDownload / File: emd_42490.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map filtered by estimated local resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.67 Å/pix.
x 352 pix.
= 234.08 Å		0.67 Å/pix.
x 352 pix.
= 234.08 Å		0.67 Å/pix.
x 352 pix.
= 234.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.665 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.5923674 - 0.9146393
Average (Standard dev.)0.00057563087 (±0.020335132)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 234.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : flavin monooxygenase asymmetric trimer

EntireName: flavin monooxygenase asymmetric trimer
Components
  • Complex: flavin monooxygenase asymmetric trimer
    • Protein or peptide: Flavin monooxygenase
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: pyridine-2,6-diol
  • Ligand: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL

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Supramolecule #1: flavin monooxygenase asymmetric trimer

SupramoleculeName: flavin monooxygenase asymmetric trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Neobacillus niacini (bacteria)

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Macromolecule #1: Flavin monooxygenase

MacromoleculeName: Flavin monooxygenase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Neobacillus niacini (bacteria)
Molecular weightTheoretical: 51.924461 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGENLYFQG HMEELIKEVQ SDVCIVGAGP AGMLLGLLLA KQGLEVIVLE QNGDFHREYR GEITQPRFVQ LMKQLNLLD YIESNSHVKI PEVNVFHNNV KIMQLAFNTL IDEESYCARL TQPTLLSALL DKAKKYPNFK LLFNTKVRDL L REDGKVTG ...String:
MGSSHHHHHH SSGENLYFQG HMEELIKEVQ SDVCIVGAGP AGMLLGLLLA KQGLEVIVLE QNGDFHREYR GEITQPRFVQ LMKQLNLLD YIESNSHVKI PEVNVFHNNV KIMQLAFNTL IDEESYCARL TQPTLLSALL DKAKKYPNFK LLFNTKVRDL L REDGKVTG VYAVAKPGEQ INFTEDEVFE GNLNIKSRVT VGVDGRNSTM EKLGNFELEL DYYDNDLLWF SFEKPESWDY NI YHFYFQK NYNYLFLPKL GGYIQCGISL TKGEYQKIKK EGIESFKEKI LEDMPILKQH FDTVTDFKSF VQLLCRMRYI KDW AKEEGC MLIGDAAHCV TPWGAVGSTL AMGTAVIAAD VIYKGFKNND LSLETLKQVQ SRRKEEVKMI QNLQLTIEKF LTRE PIKKE IAPLMFSIAT KMPDITNLYK KLFTREFPLD IDESFIFHDE LVEAN

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Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 3 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #3: pyridine-2,6-diol

MacromoleculeName: pyridine-2,6-diol / type: ligand / ID: 3 / Number of copies: 3 / Formula: WTQ
Molecular weightTheoretical: 111.099 Da

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Macromolecule #4: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL

MacromoleculeName: 1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL
type: ligand / ID: 4 / Number of copies: 3 / Formula: DR9
Molecular weightTheoretical: 746.991 Da
Chemical component information

ChemComp-DR9:
1-CIS-9-OCTADECANOYL-2-CIS-9-HEXADECANOYL PHOSPHATIDYL GLYCEROL

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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

Concentration.8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
50.0 mMsodium chlorideNaCl

Details: 20 mM Tris pH 8 50 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 7048 / Average electron dose: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 130000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3353462
Startup modelType of model: OTHER / Details: copublished cryo-EM map without (2,6)DHP bound
Final reconstructionNumber classes used: 42 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.2) / Number images used: 1454164
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0.2)
Final 3D classificationNumber classes: 120 / Software - Name: cryoSPARC (ver. 4.0.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8urc


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-8urd:
Bacillus niacini flavin monooxygenase with bound (2,6)DHP

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