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

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Basic information

Entry
Database: PDB / ID: 3ute
TitleCrystal structure of Aspergillus fumigatus UDP galactopyranose mutase sulfate complex
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
ACETATE ION / FLAVIN-ADENINE DINUCLEOTIDE / UDP-galactopyranose mutase
Similarity search - Component
Biological speciesAspergillus fumigatus (mold)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.35 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 1.3Feb 28, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / 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)233,44027
Polymers228,5144
Non-polymers4,92623
Water9,296516
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area17480 Å2
ΔGint-319 kcal/mol
Surface area75390 Å2
MethodPISA
Unit cell
Length a, b, c (Å)217.841, 217.841, 319.693
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Components on special symmetry positions
IDModelComponents
11B-516-

ACT

21B-516-

ACT

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Components

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Protein , 1 types, 4 molecules ABCD

#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

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Non-polymers , 5 types, 539 molecules

#2: Chemical
ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Comment: FAD*YM
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 17 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 516 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 4.79 Å3/Da / Density % sol: 74.33 %
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: APS / Beamline: 19-ID / Wavelength: 0.97915 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Aug 6, 2011 / Details: Rosenbaum-Rock vertical focusing mirror
RadiationMonochromator: Rosenbaum-Rock high-resolution double-crystal monochromator
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 2.35→50 Å / Num. obs: 167455 / % possible obs: 90.5 % / Redundancy: 5.4 % / Rmerge(I) obs: 0.086 / Χ2: 1.017 / Net I/σ(I): 8.4
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allΧ2Diffraction-ID% possible all
2.35-2.435.40.568166670.59191.1
2.43-2.535.40.427166640.593191.3
2.53-2.655.40.325166950.615191.3
2.65-2.795.40.24167850.644191.5
2.79-2.965.40.163167700.674191.3
2.96-3.195.40.111167990.811191.3
3.19-3.515.40.08167601.155190.7
3.51-4.025.40.069166941.884190
4.02-5.065.30.054166592.057189
5.06-505.40.034169621.166187.3

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Processing

Software
NameVersionClassificationNB
SCALEPACKdata scaling
PHENIX1.6.2_432refinement
PDB_EXTRACT3.1data extraction
ADSCQuantumdata collection
HKL-3000data reduction
PHENIXphasing
RefinementMethod to determine structure: SAD / Resolution: 2.35→49.421 Å / Occupancy max: 1 / Occupancy min: 0 / FOM work R set: 0.8482 / SU ML: 0.33 / σ(F): 0 / Phase error: 21.91 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2183 8395 5.02 %random
Rwork0.1881 ---
obs0.1896 167290 90.37 %-
Solvent computationShrinkage radii: 0.95 Å / VDW probe radii: 1.2 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 31.152 Å2 / ksol: 0.354 e/Å3
Displacement parametersBiso max: 144.47 Å2 / Biso mean: 34.984 Å2 / Biso min: 7.15 Å2
Baniso -1Baniso -2Baniso -3
1--5.6926 Å20 Å20 Å2
2---5.6926 Å2-0 Å2
3---11.3853 Å2
Refinement stepCycle: LAST / Resolution: 2.35→49.421 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15569 0 307 516 16392
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00716302
X-RAY DIFFRACTIONf_angle_d1.06422277
X-RAY DIFFRACTIONf_chiral_restr0.0682435
X-RAY DIFFRACTIONf_plane_restr0.0052870
X-RAY DIFFRACTIONf_dihedral_angle_d14.8375969
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 (%)
2.35-2.43070.29748050.2486156961650190
2.4307-2.5280.28018410.2198158361667791
2.528-2.6430.25158620.2079158381670091
2.643-2.78240.24198190.2015159751679491
2.7824-2.95670.24527990.2054159611676091
2.9567-3.18490.25198660.2146159411680791
3.1849-3.50540.2418670.2126158901675791
3.5054-4.01240.20927980.1791158831668190
4.0124-5.05440.16178610.1374158191668089
5.0544-49.43220.18958770.1769160561693387
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)
10.67660.0016-0.2690.0759-0.0280.23610.02120.1918-0.049-0.0033-0.0226-0.0769-0.0211-0.02940.00390.05750.04730.02510.1926-0.0290.122471.423577.7283159.3665
20.2792-0.25860.01210.4220.01980.2317-0.0788-0.0460.04790.09870.04130.062-0.0023-0.0020.00820.08580.04770.02230.0724-0.02230.100624.4064103.2378211.9273
30.87210.1761-0.08520.15430.06940.324-0.03260.3585-0.1832-0.0010.06990.09040.1009-0.0236-0.0618-0.03880.0339-0.07170.2199-0.10250.055925.5668.0432149.5188
40.3171-0.25460.02970.68670.06590.1972-0.0934-0.0622-0.12560.22940.11710.1280.0234-0.0074-0.00670.17090.08630.05310.11840.07910.131241.926859.7539222.1384
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 - 506
4X-RAY DIFFRACTION4chain DD3 - 506

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