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- EMDB-33113: Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE -

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Basic information

Entry
Database: EMDB / ID: EMD-33113
TitlePhotobacterium phosphoreum fatty acid reductase complex LuxC-LuxE
Map dataEM map of Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE
Sample
  • Complex: fatty acid reductase complex
    • Protein or peptide: LuxE
    • Protein or peptide: Long-chain acyl-protein thioester reductase
Keywordsfatty acid reductase / acyl-protein synthetase / acyl-CoA reductase / bacterial bioluminescenc / LUMINESCENT PROTEIN
Function / homology
Function and homology information


long-chain acyl-protein thioester reductase / long-chain fatty acid--protein ligase activity / long-chain-fatty-acyl-CoA reductase activity / acyl-CoA dehydrogenase activity / bioluminescence
Similarity search - Function
Acyl-protein synthetase, LuxE / Long-chain-fatty-acyl-CoA reductase, LuxC / Acyl-protein synthetase, LuxE, bacterial / Acyl-protein synthetase, LuxE / Acyl-CoA reductase (LuxC) / ANL, N-terminal domain / Aldehyde dehydrogenase, N-terminal / Aldehyde dehydrogenase, C-terminal / Aldehyde/histidinol dehydrogenase
Similarity search - Domain/homology
LuxE / Long-chain acyl-protein thioester reductase
Similarity search - Component
Biological speciesPhotobacterium phosphoreum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsTian Q / Huo Y / Wang L
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2022
Title: Cryo-EM structure of the fatty acid reductase LuxC-LuxE complex provides insights into bacterial bioluminescence.
Authors: Qingwei Tian / Jingting Wu / Haifeng Xu / Zhangli Hu / Yangao Huo / Liyan Wang /
Abstract: The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying ...The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.
History
DepositionMar 23, 2022-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33113.png

Downloads & links

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Map

FileDownload / File: emd_33113.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE
Voxel sizeX=Y=Z: 0.833 Å
Density
Contour LevelBy AUTHOR: 0.0178
Minimum - Maximum-0.0552294 - 0.10389767
Average (Standard dev.)-0.000051212057 (±0.0036158962)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 283.22 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EM 2 half map of Photobacterium phosphoreum fatty...

Fileemd_33113_half_map_1.map
AnnotationEM 2 half map of Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map of Photobacterium phosphoreum fatty acid...

Fileemd_33113_half_map_2.map
AnnotationEM half map of Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : fatty acid reductase complex

EntireName: fatty acid reductase complex
Components
  • Complex: fatty acid reductase complex
    • Protein or peptide: LuxE
    • Protein or peptide: Long-chain acyl-protein thioester reductase

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Supramolecule #1: fatty acid reductase complex

SupramoleculeName: fatty acid reductase complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Photobacterium phosphoreum (bacteria)

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Macromolecule #1: LuxE

MacromoleculeName: LuxE / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Photobacterium phosphoreum (bacteria)
Molecular weightTheoretical: 42.934445 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTITLDICEK DIIVSTEIDD IIFTSSPLDI TYDEQERIKH KLILESFRYH YNNNEDYKSF CNTQGVDENI SSLDDIPVFP TSMFKYAKI CTADESNIEN WFTSSGTSGV KSHIARDRVS IERLLGSVNY GMKYLGSFHE NQLELVNMGP DRFNAKNVWF K YVMSLVEL ...String:
MTITLDICEK DIIVSTEIDD IIFTSSPLDI TYDEQERIKH KLILESFRYH YNNNEDYKSF CNTQGVDENI SSLDDIPVFP TSMFKYAKI CTADESNIEN WFTSSGTSGV KSHIARDRVS IERLLGSVNY GMKYLGSFHE NQLELVNMGP DRFNAKNVWF K YVMSLVEL LYPTTFTVNN DEIDFELTIK SLKEIYNKGK GICLIGPPYF IYLLCQYMKE NDIEFNAGNR IFIITGGGWK TK QKQALNR QDFNQLLMET FHLAHESQIR DTFNQVELNT CFFEDNRQRK HVPPWVYARA LDPVTLKPVE DGQEGLISYM DAS STSYPT FIVTDDIGII HTIKAPDPHQ GTTIDIVRRL NTREQKGCSL SMSSGLK

UniProtKB: LuxE

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Macromolecule #2: Long-chain acyl-protein thioester reductase

MacromoleculeName: Long-chain acyl-protein thioester reductase / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: long-chain acyl-protein thioester reductase
Source (natural)Organism: Photobacterium phosphoreum (bacteria)
Molecular weightTheoretical: 54.067164 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: IKKIPMIIGG AERDTSEHEY RELTLNSYKV SIPIINQDDV EAIKSQSVEN NLNINQIVNF LYTVGQKWKS ENYSRRLTYI RDLVRFLGY SPEMAKLEAN WISMILSSKS ALYDIVETEL GSRHIVDEWL PQGDCYVKAM PKGKSVHLLA GNVPLSGVTS I IRAILTKN ...String:
IKKIPMIIGG AERDTSEHEY RELTLNSYKV SIPIINQDDV EAIKSQSVEN NLNINQIVNF LYTVGQKWKS ENYSRRLTYI RDLVRFLGY SPEMAKLEAN WISMILSSKS ALYDIVETEL GSRHIVDEWL PQGDCYVKAM PKGKSVHLLA GNVPLSGVTS I IRAILTKN ECIIKTSSAD PFTAIALASS FIDTDEHHPI SRSMSVMYWS HNEDIAIPQQ IMNCADVVVS WGGYDAIKWA TE HTPVNVD ILKFGPKKSI AIVDNPVDIT ASAIGVAHDI CFYDQQACFS TQDIYYIGDN IDAFFDELVE QLNLYMDILP KGD QTFDEK ASFSLIEKEC QFAKYKVEKG DNQSWLLVKS PLGSFGNQPL ARSAYIHHVS DISEITPYIE NRITQTVTVT PWES SFKYR DVLASHGAER IVESGMNNIF RVGGAHDGMR PLQRLVKYIS HERPYTYSTK DVAVKIEQTR YLEEDKFLVF VP

UniProtKB: Long-chain acyl-protein thioester reductase

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 229033
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7xc6:
Photobacterium phosphoreum fatty acid reductase complex LuxC-LuxE

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