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- EMDB-23817: Glutamate synthase, glutamate dehydrogenase counter-enzyme complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23817
TitleGlutamate synthase, glutamate dehydrogenase counter-enzyme complex
Map dataSharpened map used in model refinement.
Sample
  • Complex: GudB6-GltA2-GltB2
    • Protein or peptide: Glutamate dehydrogenase
    • Protein or peptide: Glutamate synthase (NADPH) large chain
    • Protein or peptide: Glutamate synthase (NADPH) small chain
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: FE3-S4 CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
Function / homology
Function and homology information


glutamate synthase activity / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor / glutamate biosynthetic process / glutamate dehydrogenase (NAD+) activity / amino acid metabolic process / 3 iron, 4 sulfur cluster binding / glutamine metabolic process / iron-sulfur cluster binding / nucleotide binding / metal ion binding
Similarity search - Function
Glutamate synthase, NADH/NADPH, small subunit 1 / Glutamate synthase domain / Glutamate synthase, central-N / Glutamine amidotransferases class-II / Conserved region in glutamate synthase / Glutamate synthase central domain / Glutamate synthase, alpha subunit, C-terminal / GXGXG motif / Glutamate synthase, alpha subunit, C-terminal domain superfamily / Dihydroprymidine dehydrogenase domain II ...Glutamate synthase, NADH/NADPH, small subunit 1 / Glutamate synthase domain / Glutamate synthase, central-N / Glutamine amidotransferases class-II / Conserved region in glutamate synthase / Glutamate synthase central domain / Glutamate synthase, alpha subunit, C-terminal / GXGXG motif / Glutamate synthase, alpha subunit, C-terminal domain superfamily / Dihydroprymidine dehydrogenase domain II / Dihydroprymidine dehydrogenase domain II, 4Fe-4S cluster / Glutamine amidotransferase type 2 domain profile. / Glutamine amidotransferase type 2 domain / Glutamate dehydrogenase / NAD(P) binding domain of glutamate dehydrogenase / Leu/Phe/Val dehydrogenases active site / Glu / Leu / Phe / Val dehydrogenases active site. / Alpha-helical ferredoxin / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Aminoacid dehydrogenase-like, N-terminal domain superfamily / FAD/NAD(P)-binding domain / Pyridine nucleotide-disulphide oxidoreductase / Nucleophile aminohydrolases, N-terminal / FAD/NAD(P)-binding domain superfamily / Aldolase-type TIM barrel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Glutamate dehydrogenase / Glutamate synthase (NADPH) small chain / Glutamate synthase (NADPH) large chain
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsJayaraman V / Lee DJ / Elad N / Fraser JS / Tawfik DS
Funding support Israel, United States, 2 items
OrganizationGrant numberCountry
Israel Science Foundation2575/20 Israel
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123159 United States
CitationJournal: Nat Chem Biol / Year: 2022
Title: A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.
Authors: Vijay Jayaraman / D John Lee / Nadav Elad / Shay Vimer / Michal Sharon / James S Fraser / Dan S Tawfik /
Abstract: Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from ...Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.
History
DepositionApr 10, 2021-
Header (metadata) releaseJan 5, 2022-
Map releaseJan 5, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mfm
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23817.png

Downloads & links

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Map

FileDownload / File: emd_23817.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map used in model refinement.
Voxel sizeX=Y=Z: 0.824 Å
Density
Contour LevelBy AUTHOR: 3.2 / Movie #1: 5
Minimum - Maximum-20.378754 - 47.48116
Average (Standard dev.)-1.4068342e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 421.888 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8240.8240.824
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z421.888421.888421.888
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-20.37947.481-0.000

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Supplemental data

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Additional map: Original map (not sharpened).

Fileemd_23817_additional_1.map
AnnotationOriginal map (not sharpened).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GudB6-GltA2-GltB2

EntireName: GudB6-GltA2-GltB2
Components
  • Complex: GudB6-GltA2-GltB2
    • Protein or peptide: Glutamate dehydrogenase
    • Protein or peptide: Glutamate synthase (NADPH) large chain
    • Protein or peptide: Glutamate synthase (NADPH) small chain
  • Ligand: FLAVIN MONONUCLEOTIDE
  • Ligand: FE3-S4 CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: GudB6-GltA2-GltB2

SupramoleculeName: GudB6-GltA2-GltB2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: GudB hexamer binding two GltAB heterodimers.
Source (natural)Organism: Bacillus subtilis (bacteria)
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 740 KDa

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Macromolecule #1: Glutamate dehydrogenase

MacromoleculeName: Glutamate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 47.100715 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MAADRNTGHT EEDKLDVLKS TQTVIHKALE KLGYPEEVYE LLKEPMRLLT VKIPVRMDDG SVKIFTGYRA QHNDSVGPTK GGIRFHPNV TEKEVKALSI WMSLKCGIID LPYGGGKGGI VCDPRDMSFR ELERLSRGYV RAISQIVGPT KDVPAPDVFT N SQIMAWMM ...String:
MAADRNTGHT EEDKLDVLKS TQTVIHKALE KLGYPEEVYE LLKEPMRLLT VKIPVRMDDG SVKIFTGYRA QHNDSVGPTK GGIRFHPNV TEKEVKALSI WMSLKCGIID LPYGGGKGGI VCDPRDMSFR ELERLSRGYV RAISQIVGPT KDVPAPDVFT N SQIMAWMM DEYSRIDEFN SPGFITGKPL VLGGSHGRES ATAKGVTICI KEAAKKRGID IKGARVVVQG FGNAGSYLAK FM HDAGAKV VGISDAYGGL YDPEGLDIDY LLDRRDSFGT VTKLFNDTIT NQELLELDCD ILVPAAIENQ ITEENAHNIR AKI VVEAAN GPTTLEGTKI LSDRDILLVP DVLASAGGVT VSYFEWVQNN QGFYWSEEEV EEKLEKMMVK SFNNIYEMAN NRRI DMRLA AYMVGVRKMA EASRFRGWI

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Macromolecule #2: Glutamate synthase (NADPH) large chain

MacromoleculeName: Glutamate synthase (NADPH) large chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 169.006375 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MTYNQMPKAQ GLYRPEFEHD ACGIGLYAHL KGKQTHDIVK QGLKMLCQLD HRGGQGSDPD TGDGAGLLVQ IPDAFFRKEC KNINLPEKE RYGVGMVFFS QKEDERKKIE KQINALIEQE GQVVLGWRTV PVNVGKIGTV AQKSCPFVRQ VFIGASSDLK D NLSFERKL ...String:
MTYNQMPKAQ GLYRPEFEHD ACGIGLYAHL KGKQTHDIVK QGLKMLCQLD HRGGQGSDPD TGDGAGLLVQ IPDAFFRKEC KNINLPEKE RYGVGMVFFS QKEDERKKIE KQINALIEQE GQVVLGWRTV PVNVGKIGTV AQKSCPFVRQ VFIGASSDLK D NLSFERKL YVIRKQAENW GVTEGLDFYF ASLSSQTIVY KGLLTPEQVD AFYSDLQDEA FVSAFALVHS RFSTNTFPTW ER AHPNRYL VHNGEINTLR GNINWMRARE QQFVSESFGE DLNKILPILN ADGSDSSILD NAFEFFVMAG RKPAHTAMML IPE PWTENT HMSKEKRAFY EYHSSLMEPW DGPTAISFTD GKQIGAILDR NGLRPARYYV TKDDYIIFSS EVGVIEVEQE NVLY KNRLE PGKMLLIDLE EGRIISDEEV KTQIATEYPY QKWLEEELVQ VNPDPESREE EQFSDLLTRQ KAFGYTYEDI QKYLI PVIK EGKDPLGSMG NDAPLAVLSD RAQSLFNYFK QLFAQVTNPP IDAIREQLVT STMTWLGAEG DLLHPSERNV RRIKLY TPV LSNEQFYALK TIVHPDLKSQ KIDVLFSEDL ERGLKDMFTQ AEKAISQGVS LLILSDKKMN ERLTPIPPLL AVSALHQ HL IRKGLRTKVS IIVESGEARE VHHFAALIGY GADAINPYLA YATYKQEIDE GRLDISYEEA VSKYGKSITE GVVKVMSK M GISTVQSYRG AQIFEAVGIS RDVIDRYFSG TASQLGGIDL QTIAEEAQRR HREAYQDDYS KTLEPGSDFQ WRNGGEHHA FNPKTIHTLQ WACRRNDYNL FKQYTKAADE ERIGFLRNLF AFDGNRKPLK LEEVESAESI VKRFKTGAMS FGSLSKEAHE ALAIAMNRL GGKSNSGEGG EDPKRFVPDE NGDDRRSAIK QIASGRFGVK SHYLVNADEL QIKMAQGAKP GEGGQLPGNK V YPWVADVR GSTPGVGLIS PPPHHDIYSI EDLAQLIHDL KNANRDARIS VKLVSKAGVG TIAAGVAKAT ADVIVISGYD GG TGASPKT SIKHTGLPWE LGLAEAHQTL MLNGLRDRVV LETDGKLMTG RDVVMAALLG AEEFGFATAP LVVLGCVMMR ACH LDTCPV GVATQNPELR KKFMGDPDHI VNYMLFIAEE VREYMAALGF KTFDEMIGRT DVLHVSERAK EHWKASQLDL STLL YQPEG VRTFQSPQNH KIDQSLDITT ILPAVQEAIE SGKEADISIE INNTNRVAGT ITGSEISKRY GEEGLPEDTI KLHFT GSAG QSFGAFVPKG MTLYLDGDSN DYVGKGLSGG KIIVKSSEGF NSASDDNVII GNVAFYGATS GEAYINGRAG ERFAVR NSG VNVVVEGIGD HGCEYMTGGS VVVLGDVGKN FAAGMSGGIA YVLTEDVKAF KRKCNLEMIL FESLEDEKEI QQIKAML ER HTAYTNSQKA EDLLDQWEDS VKKFVKVIPK NYKQMLASIE EQKAAGLSDE EAIMFAFEAN TKPKQNTAAS GQKQAVVQ

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Macromolecule #3: Glutamate synthase (NADPH) small chain

MacromoleculeName: Glutamate synthase (NADPH) small chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 58.010598 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)
SequenceString: MGKPTGFMEI KREKPAERDP LTRLKDWKEY SAPFSEEASK RQGARCMDCG TPFCQIGADI NGFTSGCPIY NLIPEWNGLV YRGRWKEAL ERLLKTNNFP EFTGRVCPAP CEGSCTLAIS DPAVSIKNIE RTIIDKGFEN GWIQPRIPKK RTGKKVAIVG S GPAGLASA ...String:
MGKPTGFMEI KREKPAERDP LTRLKDWKEY SAPFSEEASK RQGARCMDCG TPFCQIGADI NGFTSGCPIY NLIPEWNGLV YRGRWKEAL ERLLKTNNFP EFTGRVCPAP CEGSCTLAIS DPAVSIKNIE RTIIDKGFEN GWIQPRIPKK RTGKKVAIVG S GPAGLASA DQLNQAGHSV TVFERADRAG GLLTYGIPNM KLEKGIVERR IKLLTQEGID FVTNTEIGVD ITADELKEQF DA VILCTGA QKQRDLLIEG RDSKGVHYAM DYLTLATKSY LDSNFKDKQF IDAKGKDVIV IGGGDTGADC VATALRQKAK SVH QFGKHP KLPPARTNDN MWPEQPHVFT LEYAYEEAEA KFGRDPREYS IQTTKMVADK NGKLKELHTI QMEKVKNEHG KYEF RELPG TEKVWPAQLV FIAIGFEGTE QPLLKQFGVN SVNNKISAAY GDYQTNIDGV FAAGDARRGQ SLIVWAINEG REVAR EVDR YLMGSSVLPG SWSHPQFEKG GGSGGGSGGS AWSHPQFENK

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Macromolecule #4: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 2 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE / Flavin mononucleotide

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Macromolecule #5: FE3-S4 CLUSTER

MacromoleculeName: FE3-S4 CLUSTER / type: ligand / ID: 5 / Number of copies: 2 / Formula: F3S
Molecular weightTheoretical: 295.795 Da
Chemical component information

ChemComp-F3S:
FE3-S4 CLUSTER / Iron–sulfur cluster

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Macromolecule #6: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 6 / Number of copies: 2 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM / Flavin adenine dinucleotide

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Macromolecule #7: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 4 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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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

Concentration0.5 mg/mL
BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 47.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1353359
CTF correctionSoftware - Name: cisTEM (ver. 1.0.0beta) / Software - details: CTFFIND4 as part of cisTEM suite
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0.0beta)
Final 3D classificationNumber classes: 1 / Software - Name: cisTEM (ver. 1.0.0beta)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1.0.0beta)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0beta) / Number images used: 718672

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Atomic model buiding 1

Initial model
PDB IDChain

3k8z

chain_id: C, residue_range: 17-424

1ofd

chain_id: A

6s6t

chain_id: G
DetailsReal-space refinement involved iterative rounds of ISOLDE, Coot and PHENIX refinement. 1OFD and 6S6T were used as templates for homology modeling in SWISS-MODEL.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7mfm:
Glutamate synthase, glutamate dehydrogenase counter-enzyme complex

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