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- PDB-1huv: CRYSTAL STRUCTURE OF A SOLUBLE MUTANT OF THE MEMBRANE-ASSOCIATED ... -

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Basic information

Entry
Database: PDB / ID: 1huv
TitleCRYSTAL STRUCTURE OF A SOLUBLE MUTANT OF THE MEMBRANE-ASSOCIATED (S)-MANDELATE DEHYDROGENASE FROM PSEUDOMONAS PUTIDA AT 2.15A RESOLUTION
ComponentsL(+)-MANDELATE DEHYDROGENASE
KeywordsOXIDOREDUCTASE / TIM BARREL
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 / response to other organism / L-lactate dehydrogenase (NAD+) activity / aerobic respiration / peroxisome ...(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 / response to other organism / L-lactate dehydrogenase (NAD+) activity / aerobic respiration / 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 / Glycolate oxidase / (S)-mandelate dehydrogenase
Similarity search - Component
Biological speciesPseudomonas putida (bacteria)
Spinacia oleracea (spinach)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT, SIR / Resolution: 2.15 Å
AuthorsMathews, F.S. / Sukumar, N.
CitationJournal: 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
DepositionJan 4, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 19, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Jul 26, 2017Group: Advisory / Source and taxonomy / Category: entity_src_gen / pdbx_unobs_or_zero_occ_atoms
Revision 1.4Oct 4, 2017Group: Refinement description / Category: software
Revision 1.5Aug 9, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Remark 999Sequence chimeric mutant of (s)-mandelate dehydrogenase with residues 177-215 replaced by residues ...Sequence chimeric mutant of (s)-mandelate dehydrogenase with residues 177-215 replaced by residues 176-195 of glycolate oxidase. The authors believe SwissProt entry P20932 incorrectly lists residue 15 as ARG instead of ALA. Sequencing of MDH, as well as the original clone pSCR4, has been done a number of times including site-specific mutants and repeatedly confirmed that it is ALA 15 rather than ARG 15.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: L(+)-MANDELATE DEHYDROGENASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,8804
Polymers42,1321
Non-polymers7483
Water5,242291
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)171,52016
Polymers168,5294
Non-polymers2,99112
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 area16700 Å2
ΔGint-118 kcal/mol
Surface area51380 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)99.590, 99.590, 87.380
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number79
Space group name H-MI4
DetailsThe biological assembly is a tetramer generated from the monomer in the asymmetric unit by crystallographic 4-fold axes.

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Components

#1: Protein L(+)-MANDELATE DEHYDROGENASE / S-MANDELATE DEHYDROGENASE / MDH


Mass: 42132.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: 20 RESIDUE INSERTION FROM GLYCOLATE OXIDASE AT RESIDUE 177
Source: (gene. exp.) Pseudomonas putida (bacteria), (gene. exp.) Spinacia oleracea (spinach)
Production host: Escherichia coli (E. coli) / References: UniProt: P20932, UniProt: P05414
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P
#4: 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
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 291 / 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: 2.57 Å3/Da / Density % sol: 52.15 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 6.2
Details: MES, ammonium sulfate, ethylene glycerol, FMN, NaCl, pH 6.2, VAPOR DIFFUSION, HANGING DROP, temperature 291K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
110-15 mg/mlprotein1drop
20.2 MMES1drop
30.75 %ammonium sulfate1drop
410 %ethylene glycol1drop
50.020 mMFMN1drop
64.0 M1reservoir1mlNaCl

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Data collection

Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
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: 2.15→30 Å / Num. all: 28882 / Num. obs: 28143 / % possible obs: 98.4 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Biso Wilson estimate: 12.7 Å2 / Rsym value: 9.2 / Net I/σ(I): 21.7
Reflection shellResolution: 2.15→30 Å / Mean I/σ(I) obs: 6.3 / Rsym value: 36.2 / % possible all: 100
Reflection
*PLUS
Rmerge(I) obs: 0.092
Reflection shell
*PLUS
% possible obs: 100 % / Rmerge(I) obs: 0.362

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Processing

Software
NameVersionClassification
AMoREphasing
CNS1refinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT, SIR
Starting model: PDB ENTRY 1GOX

1gox


Resolution: 2.15→27.42 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 53281.15 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.212 2286 10 %RANDOM
Rwork0.175 ---
obs0.175 22876 98.4 %-
all-28882 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 60.08 Å2 / ksol: 0.358 e/Å3
Displacement parametersBiso mean: 23.8 Å2
Baniso -1Baniso -2Baniso -3
1-2.38 Å20 Å20 Å2
2--2.38 Å20 Å2
3----4.76 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.25 Å0.19 Å
Luzzati d res low-5 Å
Luzzati sigma a0.16 Å0.11 Å
Refinement stepCycle: LAST / Resolution: 2.15→27.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2721 0 48 291 3060
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d23.6
X-RAY DIFFRACTIONc_improper_angle_d0.94
X-RAY DIFFRACTIONc_mcbond_it1.211.5
X-RAY DIFFRACTIONc_mcangle_it1.862
X-RAY DIFFRACTIONc_scbond_it2.122
X-RAY DIFFRACTIONc_scangle_it2.912.5
LS refinement shellResolution: 2.15→2.28 Å / Rfactor Rfree error: 0.012 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.224 358 9.7 %
Rwork0.175 3345 -
obs--96 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2FMN.PARAMFMN.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER_REP.TOP
X-RAY DIFFRACTION4MES.PARAMMES.TOP
X-RAY DIFFRACTION5SO4.PARAMSO4.TOP
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
σ(F): 0 / % reflection Rfree: 10 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 23.8 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg23.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.94
X-RAY DIFFRACTIONc_mcbond_it1.5
X-RAY DIFFRACTIONc_scbond_it2
X-RAY DIFFRACTIONc_mcangle_it2
X-RAY DIFFRACTIONc_scangle_it2.5
LS refinement shell
*PLUS
Rfactor Rfree: 0.224 / % reflection Rfree: 9.7 % / Rfactor Rwork: 0.175

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