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- PDB-9krw: E. coli MaeB apo form -

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Basic information

Entry
Database: PDB / ID: 9krw
TitleE. coli MaeB apo form
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-dependent malic enzyme
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.85 Å
AuthorsSassa, M. / Yamato, H. / Tanino, H. / Fukuda, Y. / Inoue, T.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) 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
DepositionNov 28, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 3, 2025Provider: repository / Type: Initial release
Revision 1.1Dec 10, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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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
C: NADP-dependent malic enzyme
D: NADP-dependent malic enzyme
E: NADP-dependent malic enzyme
F: NADP-dependent malic enzyme
G: NADP-dependent malic enzyme
H: NADP-dependent malic enzyme
I: NADP-dependent malic enzyme
J: NADP-dependent malic enzyme
K: NADP-dependent malic enzyme
L: NADP-dependent malic enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,000,08314
Polymers1,000,03412
Non-polymers492
Water00
1
A: NADP-dependent malic enzyme
B: NADP-dependent malic enzyme
C: NADP-dependent malic enzyme
D: NADP-dependent malic enzyme
E: NADP-dependent malic enzyme
F: NADP-dependent malic enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)500,0417
Polymers500,0176
Non-polymers241
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
G: NADP-dependent malic enzyme
H: NADP-dependent malic enzyme
I: NADP-dependent malic enzyme
J: NADP-dependent malic enzyme
K: NADP-dependent malic enzyme
L: NADP-dependent malic enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)500,0417
Polymers500,0176
Non-polymers241
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)347.708, 200.289, 201.823
Angle α, β, γ (deg.)90.000, 101.353, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z

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Components

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


Mass: 83336.164 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: maeB, ypfF, b2463, JW2447 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P76558, malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)
#2: 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.44 Å3/Da / Density % sol: 64.3 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 / Details: 0.2 M Potassium chloride, 20% w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Dec 1, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 3.85→49.47 Å / Num. obs: 127049 / % possible obs: 99.1 % / Redundancy: 3.5 % / Biso Wilson estimate: 138.04 Å2 / CC1/2: 0.994 / Rmerge(I) obs: 0.14 / Rpim(I) all: 0.087 / Rrim(I) all: 0.165 / Net I/σ(I): 9.1
Reflection shellResolution: 3.85→3.92 Å / Redundancy: 3.7 % / Rmerge(I) obs: 1.315 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 6295 / CC1/2: 0.537 / Rpim(I) all: 0.798 / Rrim(I) all: 1.541 / % possible all: 99.8

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Processing

Software
NameVersionClassification
REFMACrefinement
PHENIX1.19.2_4158refinement
XDSdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6ZNJ

6znj


Resolution: 3.85→49.47 Å / SU ML: 0.5221 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.3957
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: The gap distance between residues ( F GLU 182 ) and ( F ASN 184 ) certainly seems large, but this was the limit due to the low resolution.
RfactorNum. reflection% reflection
Rfree0.24 6291 4.96 %
Rwork0.1954 120592 -
obs0.1976 126883 98.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 154.58 Å2
Refinement stepCycle: LAST / Resolution: 3.85→49.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms68717 0 2 0 68719
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002769932
X-RAY DIFFRACTIONf_angle_d0.536594839
X-RAY DIFFRACTIONf_chiral_restr0.042511003
X-RAY DIFFRACTIONf_plane_restr0.005312462
X-RAY DIFFRACTIONf_dihedral_angle_d4.17799650
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.85-3.890.37822050.363994X-RAY DIFFRACTION99.72
3.89-3.940.37372060.32974056X-RAY DIFFRACTION99.51
3.94-3.990.33582270.31184042X-RAY DIFFRACTION99.77
3.99-4.040.33371990.31344013X-RAY DIFFRACTION99.72
4.04-4.090.33091970.29914077X-RAY DIFFRACTION99.77
4.09-4.150.31922350.29413957X-RAY DIFFRACTION99.69
4.15-4.210.30822050.27354028X-RAY DIFFRACTION99.62
4.21-4.270.31292220.27094072X-RAY DIFFRACTION99.74
4.27-4.340.26661950.2584023X-RAY DIFFRACTION99.69
4.34-4.410.30582420.25573999X-RAY DIFFRACTION99.74
4.41-4.480.30981950.24264067X-RAY DIFFRACTION99.84
4.48-4.560.25862140.22984043X-RAY DIFFRACTION99.72
4.56-4.650.2772110.22134044X-RAY DIFFRACTION99.86
4.65-4.750.27071980.21994043X-RAY DIFFRACTION99.86
4.75-4.850.23082030.2094027X-RAY DIFFRACTION99.67
4.85-4.960.23292190.20474055X-RAY DIFFRACTION99.77
4.96-5.090.27072110.21864032X-RAY DIFFRACTION99.46
5.09-5.220.27181990.22114020X-RAY DIFFRACTION99.65
5.22-5.380.26992100.22794078X-RAY DIFFRACTION99.67
5.38-5.550.26362210.21584031X-RAY DIFFRACTION99.7
5.55-5.750.25012160.22424070X-RAY DIFFRACTION99.67
5.75-5.980.29992110.23664017X-RAY DIFFRACTION99.69
5.98-6.250.28092240.21194061X-RAY DIFFRACTION99.74
6.25-6.580.23722230.18824004X-RAY DIFFRACTION99.44
6.58-6.990.23222120.18724070X-RAY DIFFRACTION99.3
6.99-7.530.21571910.15824053X-RAY DIFFRACTION99.11
7.53-8.280.17942170.13284008X-RAY DIFFRACTION98.12
8.28-9.470.15792190.10873964X-RAY DIFFRACTION97.14
9.47-11.910.13281790.10113983X-RAY DIFFRACTION96.16
11.91-49.470.19461850.14513661X-RAY DIFFRACTION87.57

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