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- PDB-8q2h: beta-galactosidase from Bacillus circulans -

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Basic information

Entry
Database: PDB / ID: 8q2h
Titlebeta-galactosidase from Bacillus circulans
Componentsbeta-galactosidase
KeywordsHYDROLASE / Galactosidase / Transgalactosylation / Izoform A / Enzyme
Function / homology
Function and homology information


beta-galactosidase activity / carbohydrate metabolic process
Similarity search - Function
: / Beta-galactosidase-like, Galactose-binding domain / Bacterial Ig-like domain / Bacterial Ig-like domain (group 4) / Glycoside hydrolase family 2, domain 5 / Glycoside hydrolase family 2 C-terminal domain 5 / Domain of unknown function DUF4982 / Domain of unknown function (DUF4982) / : / Invasin/intimin cell-adhesion fragments ...: / Beta-galactosidase-like, Galactose-binding domain / Bacterial Ig-like domain / Bacterial Ig-like domain (group 4) / Glycoside hydrolase family 2, domain 5 / Glycoside hydrolase family 2 C-terminal domain 5 / Domain of unknown function DUF4982 / Domain of unknown function (DUF4982) / : / Invasin/intimin cell-adhesion fragments / 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 / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesNiallia circulans subsp. circulans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKascakova, B. / Hovorkova, M. / Petraskova, L. / Novacek, J. / Pinkas, D. / Gardian, Z. / Kren, V. / Bojarova, P. / Kuta Smatanova, I.
Funding support Czech Republic, 1items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic Czech Republic
CitationJournal: Structure / Year: 2024
Title: The variable structural flexibility of the Bacillus circulans β-galactosidase isoforms determines their unique functionalities.
Authors: Michaela Hovorková / Barbora Kaščáková / Lucie Petrásková / Petra Havlíčková / Jiří Nováček / Daniel Pinkas / Zdenko Gardian / Vladimír Křen / Pavla Bojarová / Ivana Kutá Smatanová /
Abstract: β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) ...β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as "barrier domain 8," and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, with implications for tailored GOS production.
History
DepositionAug 2, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: beta-galactosidase


Theoretical massNumber of molelcules
Total (without water)192,5841
Polymers192,5841
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein beta-galactosidase


Mass: 192584.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Niallia circulans subsp. circulans (bacteria)
References: UniProt: E5RWQ2
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: Terciary complex of beta-galactosidase isoform A / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Niallia circulans subsp. circulans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6 / Details: 50 mM sodium phosphate buffer pH 6
Buffer componentConc.: 0.18 mg/ml / Name: sodium phosphate
SpecimenConc.: 0.18 mg/ml / Embedding 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm
Image recordingAverage exposure time: 5 sec. / Electron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 265100 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00218936
ELECTRON MICROSCOPYf_angle_d0.509234180
ELECTRON MICROSCOPYf_chiral_restr0.04521448
ELECTRON MICROSCOPYf_plane_restr0.00242869
ELECTRON MICROSCOPYf_dihedral_angle_d10.49193163

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