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- PDB-9m35: E. coli MaeB acetyl-CoA bound form ME domain dimer -

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Basic information

Entry
Database: PDB / ID: 9m35
TitleE. coli MaeB acetyl-CoA bound form ME domain dimer
ComponentsNADP-dependent malic enzyme
KeywordsSTRUCTURAL PROTEIN / oxidoreductase / cryo-EM / allostery
Function / homology
Function and homology information


malolactic enzyme activity / malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) / malate dehydrogenase (decarboxylating) (NADP+) activity / oxaloacetate decarboxylase activity / malate metabolic process / acyltransferase activity / NAD binding / manganese ion binding / identical protein binding / cytosol
Similarity search - Function
NAD(P)-dependent malic enzyme / Malic enzyme, NAD-binding domain, bacterial type / : / Phosphate acetyltransferase, domain 2 / Phosphate acetyltransferase, domain 1 / Phosphate acetyl/butaryl transferase / Phosphate acetyl/butaryl transferase / Malic enzyme, conserved site / Malic enzymes signature. / Malic enzyme, N-terminal domain ...NAD(P)-dependent malic enzyme / Malic enzyme, NAD-binding domain, bacterial type / : / Phosphate acetyltransferase, domain 2 / Phosphate acetyltransferase, domain 1 / Phosphate acetyl/butaryl transferase / Phosphate acetyl/butaryl transferase / Malic enzyme, conserved site / Malic enzymes signature. / Malic enzyme, N-terminal domain / Malic enzyme, N-terminal domain / Malic enzyme, NAD-binding / Malic enzyme, N-terminal domain superfamily / Malic enzyme, N-terminal domain / Malic enzyme, NAD binding domain / Malic enzyme, NAD binding domain / Aminoacid dehydrogenase-like, N-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / NADP-dependent malic enzyme
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsSassa, M. / Yamato, H. / Tanino, H. / Fukuda, Y. / Inoue, T.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: J Biol Chem / Year: 2025
Title: Divergent acetyl-CoA binding modes mediate allosteric inhibition of bacterial hybrid-type malic enzymes.
Authors: Munetoshi Sassa / Haruka Yamato / Hiroki Tanino / Yohta Fukuda / Tsuyoshi Inoue /
Abstract: Malic enzymes (MEs) function as the bypass enzyme in the Krebs cycle and have attracted attention in a wide range of scientific and industrial fields. In contrast to eukaryotic MEs, there is ...Malic enzymes (MEs) function as the bypass enzyme in the Krebs cycle and have attracted attention in a wide range of scientific and industrial fields. In contrast to eukaryotic MEs, there is currently a lack of understanding of the structure-function relationships of prokaryotic MEs. Especially, little is known about an allosteric inhibition mechanism by an effector ligand in multi-domain MEs called hybrid-type MEs. Many bacterial hybrid-type MEs are inhibited by acetyl-CoA; however, the proposed acetyl-CoA binding site is not conserved. Here, we determined crystal and cryo-EM structures of hybrid-type MEs from Escherichia coli (EcMaeB) and Bdellovibrio bacteriovorus including complexes with acetyl-CoA. They reveal that these MaeBs have totally different acetyl-CoA binding sites and show different overall structural changes. However, the binding acetyl-CoA molecules induce identical movements of several α helices near the ligand both in EcMaeB and BbMaeB. Enzymatic assays proved that residues at the acetyl-CoA binding site are needed for inhibition. Phylogenetic analysis uncovered that EcMaeB and BbMaeB are classified into different clades of hybrid-type MEs and that the amino acid residues at the acetyl-CoA binding sites in different clades have evolved exclusively from each other. These results not only provide insights into bacterial MEs but also expand our knowledge about allosteric regulation in enzymes.
History
DepositionFeb 28, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 10, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: NADP-dependent malic enzyme
B: NADP-dependent malic enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,4556
Polymers94,9202
Non-polymers1,5354
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein NADP-dependent malic enzyme / NADP-ME


Mass: 47459.816 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: maeB, ypfF, b2463, JW2447 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P76558, malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)
#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE


Mass: 743.405 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Malic enzyme B from Escherichia coli acetyl-CoA bound form
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
11.0 mMsodium magnesiumMgCl21
23.0 mMNicotinamide adenine dinucleotide phosphateNADP+1
320 mMHEPES1
40.06 mMacetyl-coenzyme A1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 mA current / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: 3 microliters droplet, 3 seconds delay before blotting, 3 seconds blot, 0 second delay before plunging.

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10332

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 501182 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 34.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00496890
ELECTRON MICROSCOPYf_angle_d0.75049370
ELECTRON MICROSCOPYf_chiral_restr0.05341084
ELECTRON MICROSCOPYf_plane_restr0.00611214
ELECTRON MICROSCOPYf_dihedral_angle_d9.37851074

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