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- PDB-9m0e: Enhancing the synthesis efficiency of galacto-oligosaccharides of... -

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Basic information

Entry
Database: PDB / ID: 9m0e
TitleEnhancing the synthesis efficiency of galacto-oligosaccharides of a beta-galactosidase from Paenibacillus barengoltzii by engineering the active and distal sites
ComponentsBeta-galactosidase
KeywordsHYDROLASE / Beta-galactosidase / transglycosylation / GOS synthesis / cryo-EM structure
Function / homology
Function and homology information


lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding
Similarity search - Function
Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. ...Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / Glycoside hydrolase, family 2 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesPaenibacillus barengoltzii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYu, H.Y. / Wang, Y.L. / Yang, Z.S. / Liu, X. / Xin, F.J.
Funding support China, 1items
OrganizationGrant numberCountry
National Basic Research Program of China (973 Program)2023YFF1104004 China
CitationJournal: Food Chem / Year: 2025
Title: Enhancing the synthesis efficiency of galacto-oligosaccharides of a β-galactosidase from Paenibacillus barengoltzii by engineering the active and distal sites.
Authors: Haiyan Yu / Yulu Wang / Zhisen Yang / Jiabao Ying / Feifei Guan / Bolin Liu / Miao Miao / Abeer Mohamed / Xue Wei / Yuji Yang / Xin Liu / Linfeng Sun / Zhengqiang Jiang / Shaoqing Yang / Fengjiao Xin /
Abstract: Previously, a glycoside hydrolase (GH) family 2 β-galactosidase (PbBGal2A) from Paenibacillus barengoltzii is characterized for its high transglycosylation capability. Here, the cryo-electron ...Previously, a glycoside hydrolase (GH) family 2 β-galactosidase (PbBGal2A) from Paenibacillus barengoltzii is characterized for its high transglycosylation capability. Here, the cryo-electron microscopy (cryo-EM) structure of PbBGal2A was determined, revealing an enlarged acidic catalytic pocket that facilitate the binding of carbohydrate substrates. Three structure-based strategies as well as machine learning MECE platform (method for enhancing the catalytic efficiency) were employed to identify active and distal mutations with enhanced galacto-oligosaccharides (GOS) synthesis and their synergistic effects were evaluated. The best H331V mutation yielded a maximum GOS production of 76.57 % at 4 h when 35 % (w/v) of lactose was used as a substrate. Molecular dynamics (MD) simulation analysis further indicated that distal mutations increase the rigidity of the loops surrounding the catalytic pocket. This research sheds light on the structural and catalytic mechanisms of PbBGal2A, highlighting the importance of both active and distal mutations in the efficient design of customized β-galactosidases.
History
DepositionFeb 24, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-galactosidase
B: Beta-galactosidase
C: Beta-galactosidase
D: Beta-galactosidase
E: Beta-galactosidase
F: Beta-galactosidase


Theoretical massNumber of molelcules
Total (without water)694,8826
Polymers694,8826
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Beta-galactosidase / Lactase


Mass: 115813.727 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paenibacillus barengoltzii (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0C5GSQ2, beta-galactosidase
Has protein modificationN

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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: pBbGal2A / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Paenibacillus barengoltzii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.13_2998: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 275710 / Symmetry type: POINT

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