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- PDB-7wjb: Crystal structure of Lactococcus lactis subsp. cremoris GH31 alph... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7wjb | ||||||
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Title | Crystal structure of Lactococcus lactis subsp. cremoris GH31 alpha-1,3-glucosidase in complex with glucose | ||||||
![]() | Alpha-xylosidase | ||||||
![]() | HYDROLASE / nigerose / glucose / glycoside hydrolase / GH31 / carbohydrate / TIM-barrel / hexamer | ||||||
Function / homology | ![]() 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 | ||||||
Biological species | ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Ikegaya, M. / Miyazaki, T. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 178.4 KB | Display | ![]() |
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PDB format | ![]() | 132.7 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 7wj9C ![]() 7wjaSC ![]() 7wjcC ![]() 7wjdC ![]() 7wjeC ![]() 7wjfC ![]() 7wlgC S: Starting model for refinement C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Unit cell |
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Components
#1: Protein | Mass: 88028.484 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: MG1363 / Gene: llmg_1836 / Plasmid: pET28a / Production host: ![]() ![]() References: UniProt: A2RM80, Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds | ||||
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#2: Sugar | ChemComp-GLC / | ||||
#3: Sugar | ChemComp-BGC / | ||||
#4: Chemical | ChemComp-EDO / #5: Water | ChemComp-HOH / | Has ligand of interest | Y | |
-Experimental details
-Experiment
Experiment | Method: ![]() |
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Sample preparation
Crystal | Density Matthews: 3.33 Å3/Da / Density % sol: 63.06 % |
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Crystal grow | Temperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7 Details: 13% PEG 3350, 400 mM ammonium citrate buffer (pH 7.0) |
-Data collection
Diffraction | Mean temperature: 100 K / Serial crystal experiment: N |
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Diffraction source | Source: ![]() ![]() ![]() |
Detector | Type: ADSC QUANTUM 270 / Detector: CCD / Date: Mar 7, 2021 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 1 Å / Relative weight: 1 |
Reflection | Resolution: 2→50 Å / Num. obs: 81029 / % possible obs: 100 % / Redundancy: 39.6 % / CC1/2: 1 / Rmerge(I) obs: 0.091 / Net I/σ(I): 42 |
Reflection shell | Resolution: 2→2.11 Å / Redundancy: 40.3 % / Mean I/σ(I) obs: 4.7 / Num. unique obs: 11646 / CC1/2: 0.947 / % possible all: 100 |
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Processing
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Refinement | Method to determine structure: ![]() Starting model: 7WJA Resolution: 2→46.617 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.957 / WRfactor Rfree: 0.175 / WRfactor Rwork: 0.153 / SU B: 3.291 / SU ML: 0.089 / Average fsc free: 0.9311 / Average fsc work: 0.9405 / Cross valid method: FREE R-VALUE / ESU R: 0.13 / ESU R Free: 0.123 Details: Hydrogens have been added in their riding positions
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL PLUS MASK | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 38.286 Å2
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Refinement step | Cycle: LAST / Resolution: 2→46.617 Å
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Refine LS restraints |
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LS refinement shell |
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