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- PDB-8x1b: Cryo-EM structure of FpGalactosaminidase from Flavonifractor plau... -

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Basic information

Entry
Database: PDB / ID: 8x1b
TitleCryo-EM structure of FpGalactosaminidase from Flavonifractor plautii in apo state
ComponentsAlpha-galactosidase
KeywordsHYDROLASE / Flavonifractor plautii / A to O type blood / D-(+)-Galactosamine hydrochloride
Function / homologyMelibiase / : / alpha-galactosidase / alpha-galactosidase activity / Aldolase-type TIM barrel / Glycoside hydrolase superfamily / Alpha-galactosidase
Function and homology information
Biological speciesFlavonifractor plautii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsWu, G. / Han, P. / Su, C. / Zhou, M. / Luo, K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Exp Hematol Oncol / Year: 2025
Title: Structural basis of FpGalNase and its combination with FpGalNAcDeAc for efficient A-to-O blood group conversion.
Authors: Meiling Zhou / Kaishan Luo / Chao Su / Yan Sun / Zuyan Huang / Shuo Ma / Xun Gao / Jiwei Wang / Chen Zhang / Pengcheng Han / Guoqiu Wu /
Abstract: Transfusion safety and blood typing continue to present significant challenges in clinical practice, including risks of incorrect blood transfusions and blood shortages. One promising solution is the ...Transfusion safety and blood typing continue to present significant challenges in clinical practice, including risks of incorrect blood transfusions and blood shortages. One promising solution is the enzymatic conversion of all red blood cell (RBC) types into universal O-type RBCs. However, the major obstacle to this strategy is the relatively low catalytic efficiency of the enzymes involved. In this study, we investigated two enzymes from Flavonifractor plautii, N-acetylgalactosamine deacetylase (FpGalNAcDeAc) and galactosaminidase (FpGalNase), which demonstrate synergistic activity in efficiently converting A-type RBCs to O-type. We optimized treatment conditions, achieving over 99% conversion in just five minutes using phosphate buffer saline and a 16 nM enzyme concentration. Additionally, we engineered two fusion proteins, FpGalNAcDeAc-FpGalNase and FpGalNase-FpGalNAcDeAc, which showed a 28-fold increase in catalytic efficiency compared to the enzyme mixture. Using cryo-electron microscopy, we resolved the full-length structure of FpGalNase, identifying critical active site residues involved in its catalytic mechanism. This study provides essential structural and biochemical insights for clinical applications in blood group conversion, offering a promising approach for producing universal O-type RBCs.
History
DepositionNov 6, 2023Deposition site: PDBJ / Processing site: PDBJ
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-galactosidase


Theoretical massNumber of molelcules
Total (without water)72,5151
Polymers72,5151
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Alpha-galactosidase / FpGalactosaminidase


Mass: 72515.055 Da / Num. of mol.: 1 / Mutation: P566L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Flavonifractor plautii (bacteria) / Gene: ERS852411_00574 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: A0A174W4Y6
Has protein modificationY

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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: FpGalactosaminidase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Flavonifractor plautii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 673717 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035241
ELECTRON MICROSCOPYf_angle_d0.5217143
ELECTRON MICROSCOPYf_dihedral_angle_d4.052724
ELECTRON MICROSCOPYf_chiral_restr0.045753
ELECTRON MICROSCOPYf_plane_restr0.005941

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