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- PDB-20ys: R583A mutant of glycogen phosphorylase from Segatella copri in th... -

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Entry
Database: PDB / ID: 20ys
TitleR583A mutant of glycogen phosphorylase from Segatella copri in the presence of AMP
ComponentsAlpha-glucan family phosphorylase
KeywordsTRANSFERASE / glycogen phosphorylase
Function / homology
Function and homology information


glycogen phosphorylase activity / pyridoxal phosphate binding / carbohydrate metabolic process
Similarity search - Function
Alpha-glucan phosphorylase / Glycogen phosphorylase, domain of unknown function DUF3417 / : / Protein of unknown function (DUF3417) / Glycosyl transferase, family 35 / Carbohydrate phosphorylase
Similarity search - Domain/homology
Alpha-glucan family phosphorylase
Similarity search - Component
Biological speciesSegatella copri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsTakai, M. / Fukuda, Y.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Structural and mechanistic diversity of glycogen phosphorylases from gut bacteria.
Authors: Keigo Shobu / Mayu Takai / Hiroki Tanino / Yohta Fukuda / Tsuyoshi Inoue /
Abstract: Glycogen phosphorylase (GP) plays a central role in glycogen metabolism. While the structure and regulation of mammalian GPs have been extensively studied, the corresponding mechanisms in gut ...Glycogen phosphorylase (GP) plays a central role in glycogen metabolism. While the structure and regulation of mammalian GPs have been extensively studied, the corresponding mechanisms in gut bacterial GPs remain poorly understood. Here, we investigate GPs from (GP), (GP), and (GP), which represent three phylogenetic clades of GPs, using enzymatic assays, cryo-electron microscopy (cryo-EM), and X-ray crystallography. We find that GP forms a unique pentamer that undergoes adenosine monophosphate (AMP)-dependent assembly into a dimer-of-pentamer, which inhibits activity by restricting substrate access to the catalytic site. GP exists in equilibrium among monomers, dimers, and tetramers, with AMP promoting tetramer dissociation and enhancing catalytic efficiency. In contrast, GP remains predominantly monomeric and is unresponsive to AMP. These findings uncover structural and regulatory diversity among gut bacterial GPs. Notably, the oligomeric states of GPs modulate substrate accessibility and enzyme activation, suggesting a distinct mode of allosteric regulation beyond the canonical T-to-R transition model. Because bacterial GPs contribute to the generation of glucose, their regulation may influence the composition of gut-derived metabolites that affect host glucose homeostasis and insulin sensitivity. Our study provides mechanistic insight into the structural and functional diversity of gut bacterial GPs and lays a foundation for future exploration of microbiome-mediated metabolic interactions.
History
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-glucan family phosphorylase
B: Alpha-glucan family phosphorylase
D: Alpha-glucan family phosphorylase
E: Alpha-glucan family phosphorylase
C: Alpha-glucan family phosphorylase


Theoretical massNumber of molelcules
Total (without water)505,0715
Polymers505,0715
Non-polymers00
Water00
1


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

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Components

#1: Protein
Alpha-glucan family phosphorylase


Mass: 101014.227 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Segatella copri (bacteria) / Gene: glgP, DW064_01070, LYY06_13165 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0AA93BMC9
Has ligand of interestY
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: glycogen phosphorylase / Type: COMPLEX / Details: glycogen phosphorylase from Segatella copri / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Segatella copri (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HCl1
2100 mMsodium chlorideNaCl1
35 mMAMP1
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: 3 microliters droplet, 10 seconds delay before blotting, 3 seconds blot, 0 second delay before plunging

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.0particle selection
4cryoSPARC4.6.0CTF correction
7UCSF Chimeramodel fitting
9PHENIX1.19.2_4158model refinement
10cryoSPARC4.6.0initial Euler assignment
11cryoSPARC4.6.0final Euler assignment
13cryoSPARC4.6.03D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283158 / Symmetry type: POINT
Atomic model buildingPDB-ID: 9L6I

9l6i


Accession code: 9L6I / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.96 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00535734
ELECTRON MICROSCOPYf_angle_d0.65748398
ELECTRON MICROSCOPYf_dihedral_angle_d7.0054752
ELECTRON MICROSCOPYf_chiral_restr0.0455006
ELECTRON MICROSCOPYf_plane_restr0.0056238

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