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- EMDB-63544: Enhancing the synthesis efficiency of galacto-oligosaccharides of... -

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Basic information

Entry
Database: EMDB / ID: EMD-63544
TitleEnhancing the synthesis efficiency of galacto-oligosaccharides of a beta-galactosidase from Paenibacillus barengoltzii by engineering the active and distal sites
Map data
Sample
  • Complex: pBbGal2A
    • Protein or peptide: Beta-galactosidase
KeywordsBeta-galactosidase / transglycosylation / GOS synthesis / cryo-EM structure / HYDROLASE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYu HY / Wang YL / Yang ZS / Liu X / Xin FJ
Funding support China, 1 items
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, 2025-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63544.png

Downloads & links

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Map

FileDownload / File: emd_63544.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 242.4 Å		1.01 Å/pix.
x 240 pix.
= 242.4 Å		1.01 Å/pix.
x 240 pix.
= 242.4 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.01 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.874
Minimum - Maximum-1.9148728 - 3.3937237
Average (Standard dev.)0.009126492 (±0.18921989)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 242.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_63544_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63544_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : pBbGal2A

EntireName: pBbGal2A
Components
  • Complex: pBbGal2A
    • Protein or peptide: Beta-galactosidase

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Supramolecule #1: pBbGal2A

SupramoleculeName: pBbGal2A / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Paenibacillus barengoltzii (bacteria)

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Macromolecule #1: Beta-galactosidase

MacromoleculeName: Beta-galactosidase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: beta-galactosidase
Source (natural)Organism: Paenibacillus barengoltzii (bacteria)
Molecular weightTheoretical: 115.813727 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MELNREWENL SCLHIGRLPA RASYIPYESA MTARTGKRGR SPHVQTLNGN WKFRYYRSVR EVDSHFYETE TDVSGWDDLI VPSCWQTNG YDQLHYTNVN YPIPYDPPFV PDDNPAGTYV RDFNLPEAWT KKQTRIVFEG VNACFYLWVN GRFVGYSQGS R IPAEFDLT ...String:
MELNREWENL SCLHIGRLPA RASYIPYESA MTARTGKRGR SPHVQTLNGN WKFRYYRSVR EVDSHFYETE TDVSGWDDLI VPSCWQTNG YDQLHYTNVN YPIPYDPPFV PDDNPAGTYV RDFNLPEAWT KKQTRIVFEG VNACFYLWVN GRFVGYSQGS R IPAEFDLT PFVAAGRNRL AVLVLKWCDG TYLEDQDVWR FSGIYRDVYL LSRDNTHIRD VFNQPLLSDD LSEGKLRSEI ET TGSLTIQ AELRDPAGKL IGQKEAQIDG KGAMELDVPQ PQLWNAEQPR LYELILTAGQ EVLRFRVGFK KVEITDGIFR ING RAVKLK GVNRHDSHPE LGQTIPVNHM IADLKLMKRH NINTIRTSHY PNDPKFLDLC DEFGFYIIDE ADLECHGVTR TGEL EAGAF HKLSNNPDWK EAFVERAVRM VERDKNHASV IIWSMGNESG YGDNHIAMAE WTKARDASRL VHYEGADPRY YSNSD TSCL DLDSRMYPSV KEIERYALDE NSTKPLFLCE YSHAMGNGPG DLQDYWNVIY RYPKLMGGCV WEWCDHGIAA ETPDGQ RYY AYGGDFGDQP NDRNFCIDGL VFPDRRPHTG LLELKQVIAP VLIEAEDVAQ GRFRVLNRYD FSNLSHLAVS WKLEQEG DV LQQGRSGLLT AAPGETEIIS LPYDLTVAQE EGTGPLTLTC SVRQQLDTPW AEEGYEIAFY QFELPGATSA LAHEKLQS E EYAGFMTIDE QDGMLTVRGF DFEHVFDLKK GMPQQVSKHG VPLLASLARF NIWRAPMDND MNIRKEWEAA GLDHAAMKV YRSHWEQKPD ASVEIHVDFS LASYIFEPFV RGNAVWTVGV SGEIQLKVHA EVRENLPFLP RFGLELTMPK GTEEIEYYGY GPHESYIDK RASVRKGKYL LSVDDMFENY VMPQETGSRY GTEWAIASTV QGMGLKFTAA QPFSFQALHY TAEDLTAAQH T YELKRRPE TIVTLDYQMS GTGSGSCGPQ LAEPYRFTEK SFDFELTIQP IFKEEE

UniProtKB: Beta-galactosidase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 275710
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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