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- PDB-7wlg: Cryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus ... -

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Basic information

Entry
Database: PDB / ID: 7wlg
TitleCryo-EM structure of GH31 alpha-1,3-glucosidase from Lactococcus lactis subsp. cremoris
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
Biological speciesLactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.73 Å
AuthorsIkegaya, M. / Moriya, T. / Adachi, N. / Kawasaki, M. / Park, E.Y. / Miyazaki, T.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
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 13, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-xylosidase
B: Alpha-xylosidase
C: Alpha-xylosidase
D: Alpha-xylosidase
E: Alpha-xylosidase
F: Alpha-xylosidase


Theoretical massNumber of molelcules
Total (without water)528,1716
Polymers528,1716
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14320 Å2
ΔGint-58 kcal/mol
Surface area171880 Å2

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Components

#1: Protein
Alpha-xylosidase


Mass: 88028.484 Da / Num. of mol.: 6
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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: LlGH31_u1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.516 MDa / Experimental value: NO
Source (natural)Organism: Lactococcus lactis subsp. cremoris MG1363 (lactic acid bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28a
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaClSodium chloride1
220 mMsodium dihydrogen phosphateNaH2PO41
SpecimenConc.: 0.58 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 5 seconds (blot force 15)

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 65.95 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 995
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.7.6particle selectionThe general model for cryo images (neural network denoised with JANNI) is used for fully automatic particle picking
2EPUimage acquisition
4Gctf1.18CTF correctionGCTF_v1.18_sm30-7a5_cu10.1
10RELION3.1.2initial Euler assignment
11RELION3.1.2final Euler assignment
12RELION3.1.2classification
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 267294
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 44606 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00236930
ELECTRON MICROSCOPYf_angle_d0.4650022
ELECTRON MICROSCOPYf_dihedral_angle_d4.1074758
ELECTRON MICROSCOPYf_chiral_restr0.0425166
ELECTRON MICROSCOPYf_plane_restr0.0056474

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