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- PDB-8z2a: Crystal structure of Aspergillus terreus glutamate dehydrogenase ... -

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Basic information

Entry
Database: PDB / ID: 8z2a
TitleCrystal structure of Aspergillus terreus glutamate dehydrogenase (AtGDH) with sequential mutations
ComponentsGlutamate dehydrogenase
KeywordsOXIDOREDUCTASE / glutamate dehydrogenase / allostery / cooperativity / Aspergillus / cryo-EM / domain dynamics
Function / homology
Function and homology information


glutamate biosynthetic process / glutamate dehydrogenase (NADP+) activity / nucleotide binding / cytosol
Similarity search - Function
: / 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 / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal ...: / 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 / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Aminoacid dehydrogenase-like, N-terminal domain superfamily / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Glutamate dehydrogenase
Similarity search - Component
Biological speciesAspergillus terreus (mold)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsGodsora, B.K.J. / Bhaumik, P.
Funding support India, 1items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB)CRG/2021/002404 India
CitationJournal: Protein Sci / Year: 2025
Title: Conformational flexibility associated with remote residues regulates the kinetic properties of glutamate dehydrogenase.
Authors: Barsa Kanchan Jyotshna Godsora / Parijat Das / Prasoon Kumar Mishra / Anjali Sairaman / Sandip Kaledhonkar / Narayan S Punekar / Prasenjit Bhaumik /
Abstract: Glutamate dehydrogenase (GDH) is a pivotal metabolic enzyme in all living organisms, and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric ...Glutamate dehydrogenase (GDH) is a pivotal metabolic enzyme in all living organisms, and some of the GDHs exhibit substrate-dependent homotropic cooperativity. However, the mode of allosteric communication during the homotropic effect in GDHs remains poorly understood. In this study, we examined two homologous GDHs, Aspergillus niger GDH (AnGDH) and Aspergillus terreus GDH (AtGDH), with differing substrate utilization kinetics to uncover the factors driving their distinct behavior. We report the crystal structures and first-ever cryo-EM structures of apo- AtGDH and AnGDH that captured arrays of conformational ensembles. A wider mouth opening (~ 21 Å) is observed for the cooperative AnGDH as compared to the non-cooperative AtGDH (~17 Å) in their apo states. A network of interactions related to the substitutions in Domain II influence structural flexibility in these GDHs. Remarkably, we have identified a distant substitution (R246 to S) in Domain II, as a part of this network, which can impact the mouth opening and converts non-cooperative AtGDH into a cooperative enzyme. Our study demonstrates that remote residues can influence structural and kinetic properties in homologous GDHs.
History
DepositionApr 12, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutamate dehydrogenase
B: Glutamate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)97,7482
Polymers97,7482
Non-polymers00
Water25214
1
A: Glutamate dehydrogenase
B: Glutamate dehydrogenase

A: Glutamate dehydrogenase
B: Glutamate dehydrogenase

A: Glutamate dehydrogenase
B: Glutamate dehydrogenase


Theoretical massNumber of molelcules
Total (without water)293,2446
Polymers293,2446
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation8_555-z,x+1/2,-y+1/21
crystal symmetry operation11_455y-1/2,-z+1/2,-x1
Buried area23560 Å2
ΔGint-10 kcal/mol
Surface area91130 Å2
MethodPISA
Unit cell
Length a, b, c (Å)143.590, 143.590, 143.590
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number198
Space group name H-MP213
Space group name HallP2ac2ab3

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Components

#1: Protein Glutamate dehydrogenase


Mass: 48873.930 Da / Num. of mol.: 2 / Mutation: R246S,K260N,D261G,K264E,D265G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus terreus (mold) / Gene: gdhA, ATEIFO6365_0005079300 / Production host: Escherichia coli (E. coli) / References: UniProt: T2D1F5
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O
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: 2.5 Å3/Da / Density % sol: 50.8 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / Details: 0.2 M Sodium thiocyanate, 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: RRCAT INDUS-2 / Beamline: PX-BL21 / Wavelength: 0.9794 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Feb 26, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9794 Å / Relative weight: 1
Reflection twin
TypeCrystal-IDIDOperatorDomain-IDFraction
pseudo-merohedral11H, K, L10.5708
pseudo-merohedral22-K, -H, -L20.4292
ReflectionResolution: 3.1→20 Å / Num. obs: 18106 / % possible obs: 99.5 % / Redundancy: 20 % / Biso Wilson estimate: 69.29 Å2 / CC1/2: 0.99 / Net I/σ(I): 14.2
Reflection shellResolution: 3.1→3.3 Å / Num. unique obs: 3057 / CC1/2: 0.8

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
PDB-REDOrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7ECR

7ecr


Resolution: 3.1→19.91 Å / SU ML: 0.4139 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.4407
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2706 906 5 %
Rwork0.2003 17197 -
obs0.2038 18103 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 61.41 Å2
Refinement stepCycle: LAST / Resolution: 3.1→19.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6882 0 0 14 6896
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01357067
X-RAY DIFFRACTIONf_angle_d1.36349571
X-RAY DIFFRACTIONf_chiral_restr0.0821035
X-RAY DIFFRACTIONf_plane_restr0.01361268
X-RAY DIFFRACTIONf_dihedral_angle_d7.6789989
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.290.35171500.26822838X-RAY DIFFRACTION100
3.29-3.550.29551480.24512821X-RAY DIFFRACTION99.83
3.55-3.90.30731500.22622849X-RAY DIFFRACTION99.87
3.9-4.460.25831500.1932847X-RAY DIFFRACTION99.9
4.46-5.60.27281520.19082879X-RAY DIFFRACTION100
5.6-19.910.22651560.16452963X-RAY DIFFRACTION100

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