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- PDB-9e6m: Crystal structure of the G200R mutant from the maize chloroplasti... -

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Basic information

Entry
Database: PDB / ID: 9e6m
TitleCrystal structure of the G200R mutant from the maize chloroplastic photosynthetic NADP(+)-dependent malic enzyme
ComponentsMalic enzyme
KeywordsPHOTOSYNTHESIS / malic enzyme / oxidative decarboxylase / NADP-dependent C4 photosynthesis
Function / homology
Function and homology information


malate dehydrogenase (decarboxylating) (NADP+) activity / malate metabolic process / chloroplast / NAD binding / metal ion binding
Similarity search - Function
Malic oxidoreductase / 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 ...Malic oxidoreductase / 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
PYRUVIC ACID / Malic enzyme
Similarity search - Component
Biological speciesZea mays (maize)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.7 Å
AuthorsKlinke, S. / Schneberger, N. / Boehm, J.M. / Willms, S. / Hagelueken, G. / Geyer, M. / Maurino, V. / Alvarez, C.E.
Funding support Germany, Argentina, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)Grant MA2379/20-1 to VM Germany
Agencia Nacional de Promocion Cientifica y Tecnologica (FONCYT)PICT-2019-00079 Argentina
CitationJournal: Mol Biol Evol / Year: 2026
Title: A milestone in C4 carbon concentration mechanism evolution: structural remodeling of NADP-malic enzyme in Poaceae.
Authors: Jonas M Böhm / Simone Willms / Oja Ferrao / Martin Buitrago-Arango / Meike Hüdig / Gereon Poschmann / Nazanin Fazelnia / Luitgard Nagel-Steger / Sebastián Klinke / Athina Drakonaki / ...Authors: Jonas M Böhm / Simone Willms / Oja Ferrao / Martin Buitrago-Arango / Meike Hüdig / Gereon Poschmann / Nazanin Fazelnia / Luitgard Nagel-Steger / Sebastián Klinke / Athina Drakonaki / Christos Gatsogiannis / Marcos A Tronconi / Clarisa E Alvarez / Veronica G Maurino /
Abstract: The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme ...The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum-members of the same C4 lineage-underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.
History
DepositionOct 30, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2026Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Malic enzyme
B: Malic enzyme
C: Malic enzyme
D: Malic enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)255,50610
Polymers254,9784
Non-polymers5286
Water52229
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area19370 Å2
ΔGint-72 kcal/mol
Surface area76820 Å2
MethodPISA
Unit cell
Length a, b, c (Å)98.679, 124.161, 189.472
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
33
44
55
66
/ NCS ensembles :
ID
1
2
3
4
5
6

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Components

#1: Protein
Malic enzyme


Mass: 63744.512 Da / Num. of mol.: 4 / Mutation: G200R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zea mays (maize) / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B4F8P6
#2: Chemical
ChemComp-PYR / PYRUVIC ACID


Mass: 88.062 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C3H4O3 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 29 / Source method: isolated from a natural source / Formula: H2O
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: 2.28 Å3/Da / Density % sol: 45.96 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 4.6
Details: 2.0 M ammonium sulfate, 0.1 M sodium acetate/acetic acid buffer, pH 4.6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.97623 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 15, 2024
RadiationMonochromator: Oxford-FMB / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97623 Å / Relative weight: 1
ReflectionResolution: 2.7→124.16 Å / Num. obs: 63889 / % possible obs: 99.1 % / Redundancy: 5.3 % / Biso Wilson estimate: 50 Å2 / CC1/2: 0.993 / Rmerge(I) obs: 0.19 / Rpim(I) all: 0.092 / Rrim(I) all: 0.213 / Net I/σ(I): 5.3
Reflection shellResolution: 2.7→2.77 Å / Redundancy: 5.5 % / Rmerge(I) obs: 1.687 / Mean I/σ(I) obs: 1.1 / Num. unique obs: 4473 / CC1/2: 0.62 / Rpim(I) all: 0.78 / Rrim(I) all: 1.865 / % possible all: 99.8

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
MxCuBEdata collection
XDSdata reduction
Aimlessdata scaling
MrBUMPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.7→50.01 Å / Cor.coef. Fo:Fc: 0.947 / Cor.coef. Fo:Fc free: 0.915 / SU B: 26.425 / SU ML: 0.48 / Cross valid method: THROUGHOUT / ESU R Free: 0.439 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.29617 3227 5.1 %RANDOM
Rwork0.23895 ---
obs0.24182 60592 98.74 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 61.416 Å2
Baniso -1Baniso -2Baniso -3
1--6.49 Å20 Å2-0 Å2
2--9.77 Å20 Å2
3----3.27 Å2
Refinement stepCycle: 1 / Resolution: 2.7→50.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17433 0 36 29 17498
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.01317851
X-RAY DIFFRACTIONr_bond_other_d0.0010.01716718
X-RAY DIFFRACTIONr_angle_refined_deg1.7071.64324206
X-RAY DIFFRACTIONr_angle_other_deg1.2531.56938851
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.98152231
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.28122.761873
X-RAY DIFFRACTIONr_dihedral_angle_3_deg20.407153044
X-RAY DIFFRACTIONr_dihedral_angle_4_deg21.3831592
X-RAY DIFFRACTIONr_chiral_restr0.0770.22311
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.0219933
X-RAY DIFFRACTIONr_gen_planes_other0.0020.023627
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it6.336.2728936
X-RAY DIFFRACTIONr_mcbond_other6.336.2718935
X-RAY DIFFRACTIONr_mcangle_it9.2799.40911163
X-RAY DIFFRACTIONr_mcangle_other9.2789.4111164
X-RAY DIFFRACTIONr_scbond_it7.1196.8968915
X-RAY DIFFRACTIONr_scbond_other7.1196.8968916
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other10.74310.08913044
X-RAY DIFFRACTIONr_long_range_B_refined13.76173.63619478
X-RAY DIFFRACTIONr_long_range_B_other13.76173.63819479
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A169650.12
12B169650.12
21A172220.11
22C172220.11
31A176060.11
32D176060.11
41B171760.11
42C171760.11
51B170350.12
52D170350.12
61C171770.11
62D171770.11
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.432 286 -
Rwork0.421 4371 -
obs--99.72 %

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