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- PDB-2a85: Crystal Structure of the G81A mutant of the Active Chimera of (S)... -

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Entry
Database: PDB / ID: 2a85
TitleCrystal Structure of the G81A mutant of the Active Chimera of (S)-Mandelate Dehydrogenase in complex with its substrate 2-hydroxyoctanoate
ComponentsL(+)-mandelate dehydrogenase
KeywordsOXIDOREDUCTASE / TIM BARREL / Hydroxy acid oxidizing enzyme
Function / homology
Function and homology information


(S)-mandelate dehydrogenase / (S)-mandelate dehydrogenase activity / oxidative photosynthetic carbon pathway / (S)-2-hydroxy-acid oxidase / (S)-2-hydroxy-acid oxidase activity / mandelate catabolic process / lactate oxidation / fatty acid alpha-oxidation / response to other organism / L-lactate dehydrogenase activity ...(S)-mandelate dehydrogenase / (S)-mandelate dehydrogenase activity / oxidative photosynthetic carbon pathway / (S)-2-hydroxy-acid oxidase / (S)-2-hydroxy-acid oxidase activity / mandelate catabolic process / lactate oxidation / fatty acid alpha-oxidation / response to other organism / L-lactate dehydrogenase activity / peroxisome / FMN binding / plasma membrane
Similarity search - Function
Alpha-hydroxy acid dehydrogenase, FMN-dependent / FMN-dependent alpha-hydroxy acid dehydrogenase, active site / FMN hydroxy acid dehydrogenase domain / FMN-dependent alpha-hydroxy acid dehydrogenases active site. / FMN-dependent alpha-hydroxy acid dehydrogenase domain profile. / FMN-dependent dehydrogenase / FMN-dependent dehydrogenase / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel ...Alpha-hydroxy acid dehydrogenase, FMN-dependent / FMN-dependent alpha-hydroxy acid dehydrogenase, active site / FMN hydroxy acid dehydrogenase domain / FMN-dependent alpha-hydroxy acid dehydrogenases active site. / FMN-dependent alpha-hydroxy acid dehydrogenase domain profile. / FMN-dependent dehydrogenase / FMN-dependent dehydrogenase / Aldolase class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
FLAVIN MONONUCLEOTIDE / (2S)-2-HYDROXYOCTANOIC ACID / Glycolate oxidase / (S)-mandelate dehydrogenase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
Spinacia oleracea (spinach)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsSukumar, N. / Xu, Y. / Mitra, B. / Mathews, F.S.
Citation
Journal: Acta Crystallogr.,Sect.D / Year: 2009
Title: Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.
Authors: Sukumar, N. / Dewanti, A. / Merli, A. / Rossi, G.L. / Mitra, B. / Mathews, F.S.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: High resolution structures of an oxidized and reduced flavoprotein. The water switch in a soluble form of (S)-mandelate dehydrogenase
Authors: Sukumar, N. / Dewanti, A.R. / Mitra, B. / Mathews, F.S.
#2: Journal: Biochemistry / Year: 2001
Title: Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase
Authors: Sukumar, N. / Xu, Y. / Gatti, D.L. / Mitra, B. / Mathews, F.S.
History
DepositionJul 7, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 11, 2006Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Aug 23, 2017Group: Advisory / Refinement description / Source and taxonomy
Category: entity_src_gen / pdbx_unobs_or_zero_occ_atoms / software
Revision 1.4Oct 20, 2021Group: Advisory / Database references / Derived calculations
Category: database_2 / pdbx_unobs_or_zero_occ_atoms ...database_2 / pdbx_unobs_or_zero_occ_atoms / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Aug 23, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: L(+)-mandelate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,9584
Polymers42,1461
Non-polymers8123
Water2,270126
1
A: L(+)-mandelate dehydrogenase
hetero molecules

A: L(+)-mandelate dehydrogenase
hetero molecules

A: L(+)-mandelate dehydrogenase
hetero molecules

A: L(+)-mandelate dehydrogenase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,83216
Polymers168,5854
Non-polymers3,24712
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_775-x+2,-y+2,z1
crystal symmetry operation3_755-y+2,x,z1
crystal symmetry operation4_575y,-x+2,z1
Buried area17740 Å2
ΔGint-12 kcal/mol
Surface area49830 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)98.8, 98.8, 87.4
Angle α, β, γ (deg.)90.0, 90.0, 90.0
Int Tables number79
Space group name H-MI4

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Components

#1: Protein L(+)-mandelate dehydrogenase / S-mandelate dehydrogenase / MDH


Mass: 42146.297 Da / Num. of mol.: 1 / Mutation: G81A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas putida (bacteria), (gene. exp.) Spinacia oleracea (spinach)
Production host: Escherichia coli (E. coli)
References: UniProt: P20932, UniProt: P05414, Oxidoreductases
#2: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P
#3: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#4: Chemical ChemComp-HOC / (2S)-2-HYDROXYOCTANOIC ACID


Mass: 160.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H16O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 126 / Source method: isolated from a natural source / Formula: H2O

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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: 2.58 Å3/Da / Density % sol: 52.36 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop / pH: 6.2
Details: 0.2M MES, 0.75% ammonium sulfate, 10% ethylene glycol, 20uM FMN. Crystals grown in this condition were soaked in 8mM 2-hydroxyoctanoate for 16 hours., pH 6.2, VAPOR DIFFUSION, SITTING DROP, temperature 291K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.5→50 Å / Num. obs: 14637 / Redundancy: 5.7 % / Rmerge(I) obs: 0.118
Reflection shellResolution: 2.5→2.59 Å / Rmerge(I) obs: 0.77

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Processing

Software
NameClassification
HKL-2000data collection
SCALEPACKdata scaling
CNSrefinement
HKL-2000data reduction
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB Entry 1P4C

1p4c


Resolution: 2.5→50 Å / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflectionSelection details
Rfree0.259 680 Random
Rwork0.189 --
obs-13774 -
Displacement parametersBiso mean: 45 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.37 Å0.26 Å
Luzzati d res low-5 Å
Luzzati sigma a0.41 Å0.33 Å
Refinement stepCycle: LAST / Resolution: 2.5→50 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2748 0 54 126 2928
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d21.9
X-RAY DIFFRACTIONc_improper_angle_d0.98
LS refinement shellResolution: 2.5→2.66 Å / Rfactor Rfree error: 3.3
RfactorNum. reflection
Rfree0.312 87
Rwork0.274 -
obs-2013

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