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- PDB-7wjd: Crystal structure of Lactococcus lactis subsp. cremoris GH31 alph... -

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Basic information

Entry
Database: PDB / ID: 7wjd
TitleCrystal structure of Lactococcus lactis subsp. cremoris GH31 alpha-1,3-glucosidase mutant D394A in complex with nigerotriose
ComponentsAlpha-xylosidase
KeywordsHYDROLASE / nigerose / glucose / glycoside hydrolase / GH31 / carbohydrate / TIM-barrel / hexamer
Function / homology
Function and homology information


Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / hydrolase activity, hydrolyzing O-glycosyl compounds / carbohydrate metabolic process
Similarity search - Function
: / Glycosyl hydrolase family 31 C-terminal domain / Glycoside hydrolase family 31 / Glycosyl hydrolases family 31 TIM-barrel domain / Glycosyl hydrolase, all-beta / Glycoside hydrolase superfamily
Similarity search - Domain/homology
beta-D-glucopyranose / Alpha-xylosidase
Similarity search - Component
Biological speciesLactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsIkegaya, M. / Miyazaki, T.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)19K15748 Japan
CitationJournal: J Biol Chem / Year: 2022
Title: Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides.
Authors: Marina Ikegaya / Toshio Moriya / Naruhiko Adachi / Masato Kawasaki / Enoch Y Park / Takatsugu Miyazaki /
Abstract: Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is ...Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k/K values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.
History
DepositionJan 6, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release
Revision 1.1May 11, 2022Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Nov 29, 2023Group: Data collection / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-xylosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)88,7935
Polymers87,9841
Non-polymers8094
Water8,647480
1
A: Alpha-xylosidase
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)532,75930
Polymers527,9076
Non-polymers4,85224
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_655-y+1,x-y,z1
crystal symmetry operation3_665-x+y+1,-x+1,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/21
crystal symmetry operation11_655-x+y+1,y,-z+1/21
crystal symmetry operation12_555x,x-y,-z+1/21
Buried area34710 Å2
ΔGint20 kcal/mol
Surface area152310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)151.970, 151.970, 177.671
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322

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Components

#1: Protein Alpha-xylosidase


Mass: 87984.477 Da / Num. of mol.: 1 / Mutation: D394A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
Strain: MG1363 / Gene: llmg_1836 / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A2RM80, Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
#2: Polysaccharide alpha-D-glucopyranose-(1-3)-alpha-D-glucopyranose-(1-3)-alpha-D-glucopyranose


Type: oligosaccharide / Mass: 504.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpa1-3DGlcpa1-3DGlcpa1-ROHGlycam Condensed SequenceGMML 1.0
[][a-D-Glcp]{[(3+1)][a-D-Glcp]{[(3+1)][a-D-Glcp]{}}}LINUCSPDB-CARE
#3: Sugar ChemComp-BGC / beta-D-glucopyranose / beta-D-glucose / D-glucose / glucose / Glucose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-glucopyranoseCOMMON NAMEGMML 1.0
b-D-GlcpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 480 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 3.37 Å3/Da / Density % sol: 63.45 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 13% PEG 3350, 400 mM ammonium citrate buffer (pH 7.0), 10 mM nigerotriose

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Photon Factory / Beamline: BL-5A / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS3 S 2M / Detector: PIXEL / Date: Jun 5, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. obs: 111675 / % possible obs: 100 % / Redundancy: 19.6 % / CC1/2: 1 / Rmerge(I) obs: 0.072 / Net I/σ(I): 30.5
Reflection shellResolution: 1.8→1.9 Å / Redundancy: 20.2 % / Rmerge(I) obs: 1.505 / Mean I/σ(I) obs: 2.6 / Num. unique obs: 16068 / CC1/2: 0.841 / % possible all: 100

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Processing

Software
NameVersionClassification
REFMAC5.8.0267refinement
XDSdata reduction
SCALAdata scaling
MOLREPphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7WJA

7wja


Resolution: 1.8→47.948 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.964 / WRfactor Rfree: 0.173 / WRfactor Rwork: 0.157 / SU B: 2.236 / SU ML: 0.067 / Average fsc free: 0.9309 / Average fsc work: 0.9368 / Cross valid method: FREE R-VALUE / ESU R: 0.092 / ESU R Free: 0.088
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.1944 5487 4.916 %
Rwork0.1757 106123 -
all0.177 --
obs-111610 99.973 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL PLUS MASK
Displacement parametersBiso mean: 35.264 Å2
Baniso -1Baniso -2Baniso -3
1-0.483 Å20.242 Å20 Å2
2--0.483 Å2-0 Å2
3----1.568 Å2
Refinement stepCycle: LAST / Resolution: 1.8→47.948 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5988 0 54 480 6522
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0136234
X-RAY DIFFRACTIONr_bond_other_d0.0010.0165658
X-RAY DIFFRACTIONr_angle_refined_deg1.5951.6528447
X-RAY DIFFRACTIONr_angle_other_deg1.4211.58813077
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.4035732
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.5623.476351
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.005151052
X-RAY DIFFRACTIONr_dihedral_angle_4_deg12.2211527
X-RAY DIFFRACTIONr_chiral_restr0.0810.2787
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.027568
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021514
X-RAY DIFFRACTIONr_nbd_refined0.2040.21162
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1730.25331
X-RAY DIFFRACTIONr_nbtor_refined0.1780.22994
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0770.22837
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1260.2417
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.030.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.1740.213
X-RAY DIFFRACTIONr_nbd_other0.1820.287
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.0690.225
X-RAY DIFFRACTIONr_mcbond_it2.7263.5442922
X-RAY DIFFRACTIONr_mcbond_other2.7213.5432921
X-RAY DIFFRACTIONr_mcangle_it3.3155.3013652
X-RAY DIFFRACTIONr_mcangle_other3.3165.3023653
X-RAY DIFFRACTIONr_scbond_it3.6583.8273312
X-RAY DIFFRACTIONr_scbond_other3.6583.8273313
X-RAY DIFFRACTIONr_scangle_it5.315.5984793
X-RAY DIFFRACTIONr_scangle_other5.3095.5994794
X-RAY DIFFRACTIONr_lrange_it5.9639.9057133
X-RAY DIFFRACTIONr_lrange_other5.94139.6437037
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.8470.3054090.2747737X-RAY DIFFRACTION99.9877
1.847-1.8970.2543650.2587553X-RAY DIFFRACTION100
1.897-1.9520.2643600.2397371X-RAY DIFFRACTION99.9741
1.952-2.0120.2683270.2237165X-RAY DIFFRACTION99.9867
2.012-2.0780.2253830.2096909X-RAY DIFFRACTION99.9863
2.078-2.1510.2283260.2056707X-RAY DIFFRACTION99.9716
2.151-2.2330.2083500.1926500X-RAY DIFFRACTION99.9708
2.233-2.3240.223140.1866231X-RAY DIFFRACTION99.9847
2.324-2.4270.2283190.185999X-RAY DIFFRACTION99.9684
2.427-2.5450.2053010.1775742X-RAY DIFFRACTION100
2.545-2.6830.2042810.1775476X-RAY DIFFRACTION99.9826
2.683-2.8460.1932770.1765173X-RAY DIFFRACTION99.9817
2.846-3.0420.2242700.1784885X-RAY DIFFRACTION99.9612
3.042-3.2860.1992380.1794577X-RAY DIFFRACTION100
3.286-3.5990.1861960.1864254X-RAY DIFFRACTION100
3.599-4.0230.161900.1543858X-RAY DIFFRACTION99.9753
4.023-4.6450.1622110.1363377X-RAY DIFFRACTION99.9721
4.645-5.6870.1521700.1432913X-RAY DIFFRACTION100
5.687-8.0360.1791240.1472323X-RAY DIFFRACTION100
8.036-47.9480.157760.1651374X-RAY DIFFRACTION99.1792

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