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- PDB-3utf: Crystal structure of Aspergillus fumigatus UDP galactopyranose mu... -

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Basic information

Entry
Database: PDB / ID: 3utf
TitleCrystal structure of Aspergillus fumigatus UDP galactopyranose mutase in reduced state
ComponentsUDP-galactopyranose mutase
KeywordsISOMERASE / Nucleotide binding / Mutase / Flavin adenine dinucleotide binding
Function / homology
Function and homology information


UDP-galactopyranose mutase / UDP-galactopyranose mutase activity / cell wall organization / nucleotide binding
Similarity search - Function
NAD(P)-binding Rossmann-like domain / FAD/NAD(P)-binding domain / FAD/NAD(P)-binding domain / 3-Layer(bba) Sandwich / FAD/NAD(P)-binding domain superfamily / Alpha Beta
Similarity search - Domain/homology
DIHYDROFLAVINE-ADENINE DINUCLEOTIDE / UDP-galactopyranose mutase
Similarity search - Component
Biological speciesAspergillus fumigatus (mold)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.25 Å
AuthorsDhatwalia, R. / Singh, H. / Tanner, J.J.
CitationJournal: J Biol Chem / Year: 2012
Title: Crystal structures and small-angle x-ray scattering analysis of UDP-galactopyranose mutase from the pathogenic fungus Aspergillus fumigatus.
Authors: Richa Dhatwalia / Harkewal Singh / Michelle Oppenheimer / Dale B Karr / Jay C Nix / Pablo Sobrado / John J Tanner /
Abstract: UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. ...UDP-galactopyranose mutase (UGM) is a flavoenzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, which is a central reaction in galactofuranose biosynthesis. Galactofuranose has never been found in humans but is an essential building block of the cell wall and extracellular matrix of many bacteria, fungi, and protozoa. The importance of UGM for the viability of many pathogens and its absence in humans make UGM a potential drug target. Here we report the first crystal structures and small-angle x-ray scattering data for UGM from the fungus Aspergillus fumigatus, the causative agent of aspergillosis. The structures reveal that Aspergillus UGM has several extra secondary and tertiary structural elements that are not found in bacterial UGMs yet are important for substrate recognition and oligomerization. Small-angle x-ray scattering data show that Aspergillus UGM forms a tetramer in solution, which is unprecedented for UGMs. The binding of UDP or the substrate induces profound conformational changes in the enzyme. Two loops on opposite sides of the active site move toward each other by over 10 Å to cover the substrate and create a closed active site. The degree of substrate-induced conformational change exceeds that of bacterial UGMs and is a direct consequence of the unique quaternary structure of Aspergillus UGM. Galactopyranose binds at the re face of the FAD isoalloxazine with the anomeric carbon atom poised for nucleophilic attack by the FAD N5 atom. The structural data provide new insight into substrate recognition and the catalytic mechanism and thus will aid inhibitor design.
History
DepositionNov 25, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2012Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2012Group: Database references
Revision 1.2Apr 4, 2012Group: Database references
Revision 1.3Sep 13, 2023Group: Data collection / Database references ...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 / 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: UDP-galactopyranose mutase
B: UDP-galactopyranose mutase
C: UDP-galactopyranose mutase
D: UDP-galactopyranose mutase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)233,87431
Polymers228,5144
Non-polymers5,36027
Water14,646813
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17670 Å2
ΔGint-322 kcal/mol
Surface area75420 Å2
MethodPISA
Unit cell
Length a, b, c (Å)217.965, 217.965, 322.805
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522

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Components

#1: Protein
UDP-galactopyranose mutase


Mass: 57128.484 Da / Num. of mol.: 4 / Mutation: K344A, K345A, A429T
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aspergillus fumigatus (mold) / Gene: glf, glfA / Plasmid: pVP56K / Production host: Escherichia coli (E. coli) / References: UniProt: Q4W1X2, UDP-galactopyranose mutase
#2: Chemical
ChemComp-FDA / DIHYDROFLAVINE-ADENINE DINUCLEOTIDE


Mass: 787.566 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H35N9O15P2
#3: Chemical...
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 23 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 813 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsAUTHOR STATES THAT A429T IS AN UNINTENDED MUTATION ON THE SURFACE OF THE PROTEIN.

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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: 4.84 Å3/Da / Density % sol: 74.6 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 4.5
Details: 1.5M ammonium sulfate, 0.1M sodium acetate, 5mM L-cysteine, 0.5mM THP , pH 4.5, VAPOR DIFFUSION, SITTING DROP, temperature 298.0K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Sep 4, 2011
RadiationMonochromator: double crystal / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.25→48.243 Å / Num. all: 211192 / Num. obs: 211192 / % possible obs: 99.8 % / Redundancy: 7.4 % / Rsym value: 0.097 / Net I/σ(I): 16.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRsym value% possible all
2.25-2.377.40.5991.3227555305960.599100
2.37-2.527.50.4221.9216225289620.422100
2.52-2.697.50.32.6203883273000.3100
2.69-2.97.50.23.9190102254440.2100
2.9-3.187.40.1266.2174467234190.12699.9
3.18-3.567.40.0799.7156733212500.07999.8
3.56-4.117.30.05513.4136681187950.05599.6
4.11-5.037.20.04316.3114739159350.04399.4
5.03-7.127.20.04316.689675124600.04399.1
7.12-48.2437.20.02525.15064970310.02597.8

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Processing

Software
NameVersionClassificationNB
SCALA3.3.16data scaling
PHENIX1.6.2_432refinement
PDB_EXTRACT3.1data extraction
ADSCQuantumdata collection
XDSdata reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3UTE

3ute


Resolution: 2.25→48.243 Å / Occupancy max: 1 / Occupancy min: 0 / FOM work R set: 0.8812 / SU ML: 0.32 / σ(F): 0 / Phase error: 19.07 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2155 10571 5.01 %based on test set used for refinement of AfUGM-sulfate complex of the same enzyme
Rwork0.1902 ---
obs0.1914 211140 99.62 %-
Solvent computationShrinkage radii: 0.95 Å / VDW probe radii: 1.2 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 40.877 Å2 / ksol: 0.352 e/Å3
Displacement parametersBiso max: 110.64 Å2 / Biso mean: 30.8325 Å2 / Biso min: 10.33 Å2
Baniso -1Baniso -2Baniso -3
1--2.6975 Å20 Å20 Å2
2---2.6975 Å20 Å2
3---5.395 Å2
Refinement stepCycle: LAST / Resolution: 2.25→48.243 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15580 0 327 813 16720
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00716296
X-RAY DIFFRACTIONf_angle_d1.04422272
X-RAY DIFFRACTIONf_chiral_restr0.0672432
X-RAY DIFFRACTIONf_plane_restr0.0052874
X-RAY DIFFRACTIONf_dihedral_angle_d15.5015926
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.25-2.33040.280610610.24361984520906100
2.3304-2.42370.27859890.22761997720966100
2.4237-2.5340.251410660.20861990720973100
2.534-2.66760.23710780.19871988720965100
2.6676-2.83470.2210200.19052000821028100
2.8347-3.05360.232110590.1961998721046100
3.0536-3.36080.240110620.21332006021122100
3.3608-3.84690.227510710.20082007721148100
3.8469-4.8460.162610530.1473202212127499
4.846-48.25360.184911120.1765206002171298
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.48580.0101-0.15990.1204-0.06260.22950.00890.1275-0.02950.00530.0071-0.0515-0.0058-0.0234-0.01180.08320.03210.02910.1861-0.02870.150571.763677.9736161.0038
20.2559-0.14170.02690.3021-0.00670.2071-0.0723-0.0078-0.00910.06790.02730.06770.00310.00120.03420.12970.0430.02070.1121-0.01670.153324.4843103.2193213.6891
30.71410.0966-0.05450.18820.0690.2091-0.02360.2523-0.1199-0.01480.04430.01010.0496-0.01-0.02570.08660.0254-0.02610.2303-0.07570.1325.775968.6036150.9799
40.3243-0.25060.03370.5356-0.00330.3027-0.058-0.0221-0.09690.15990.08920.11430.0241-0.013-0.03150.1620.04870.03230.13110.06280.152242.311759.8133224.0026
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain AA3 - 507
2X-RAY DIFFRACTION2chain BB3 - 507
3X-RAY DIFFRACTION3chain CC3 - 507
4X-RAY DIFFRACTION4chain DD3 - 507

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