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- PDB-7mft: Glutamate synthase, glutamate dehydrogenase counter-enzyme comple... -

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Basic information

Entry
Database: PDB / ID: 7mft
TitleGlutamate synthase, glutamate dehydrogenase counter-enzyme complex (GudB6-GltA6-GltB6)
Components
  • (Glutamate synthase (NADPH) ...Glutamate synthase (NADPH)) x 2
  • Glutamate dehydrogenase
KeywordsCYTOSOLIC PROTEIN / Counter-enzyme complex / glutamate synthase / glutamate dehydrogenae
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. / Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Alpha-helical ferredoxin / 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
FE3-S4 CLUSTER / FLAVIN-ADENINE DINUCLEOTIDE / FLAVIN MONONUCLEOTIDE / IRON/SULFUR CLUSTER / Glutamate dehydrogenase / Glutamate synthase (NADPH) small chain / Glutamate synthase (NADPH) large chain
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJayaraman, V. / Lee, D.J. / Elad, N. / Fraser, J.S. / Tawfik, D.S.
Funding support Israel, United States, 2items
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 11, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 5, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Derived calculations
Category: citation / citation_author / pdbx_struct_oper_list
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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Assembly

Deposited unit
A: Glutamate dehydrogenase
G: Glutamate synthase (NADPH) large chain
I: Glutamate synthase (NADPH) small chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)276,3598
Polymers274,1183
Non-polymers2,2415
Water0
1
A: Glutamate dehydrogenase
G: Glutamate synthase (NADPH) large chain
I: Glutamate synthase (NADPH) small chain
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)1,658,15248
Polymers1,644,70618
Non-polymers13,44630
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: D3 (2x3 fold dihedral))

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Glutamate dehydrogenase /


Mass: 47100.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10 (bacteria)
Gene: gudB, B4122_2172, ETA10_11605, ETK61_12750, GII81_12565, SC09_Contig24orf00413
Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A0C3GZC9

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Glutamate synthase (NADPH) ... , 2 types, 2 molecules GI

#2: Protein Glutamate synthase (NADPH) large chain


Mass: 169006.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10 (bacteria)
Gene: B4417_0011 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A164XVV7
#3: Protein Glutamate synthase (NADPH) small chain


Mass: 58010.598 Da / Num. of mol.: 1 / Mutation: D78G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10 (bacteria)
Gene: B4417_0010 / Production host: Bacillus subtilis (bacteria) / References: UniProt: A0A164XVU4

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Non-polymers , 4 types, 5 molecules

#4: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P
#5: Chemical ChemComp-F3S / FE3-S4 CLUSTER / Iron–sulfur cluster


Mass: 295.795 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe3S4
#6: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN

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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: GudB-GltA-GltB / Type: COMPLEX
Details: Asymmetric unit (GudB-GltA-GltB) from a full GudB6-GltA6-GltB6 complex.
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 1.62 MDa / Experimental value: YES
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7.9
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 47.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategoryDetails
6UCSF Chimera1.15model fittingRigid-body alignment of monomers.
8ISOLDE1.1.0model refinementBackbone positioning
9PHENIX1.19.1-4122model refinementReal-space refine
10Coot0.9.3model refinementModel examination and refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 60487
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11878 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Real-space refinement involved iterative rounds of ISOLDE, Coot and PHENIX refinement. 1OFD and 6S6T were used as templates for homology modeling in SWISS-MODEL.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
13K8Z

3k8z

C117-424
21OFD

1ofd

A1
36S6T

6s6t

G1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 97.66 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00219114
ELECTRON MICROSCOPYf_angle_d0.52525844
ELECTRON MICROSCOPYf_dihedral_angle_d4.6052591
ELECTRON MICROSCOPYf_chiral_restr0.042817
ELECTRON MICROSCOPYf_plane_restr0.0043364

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