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- PDB-9o6a: CryoEM structure of EcKatG S-Trp105 at 2.22 Angstrom resolution r... -

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Basic information

Entry
Database: PDB / ID: 9o6a
TitleCryoEM structure of EcKatG S-Trp105 at 2.22 Angstrom resolution revealing an asymmetric sulfur center in O=S-Trp
ComponentsCatalase-peroxidase
KeywordsOXIDOREDUCTASE / Met-Tyr-Trp cofactor / heme-dependent enzyme / non-canonical amino acid
Function / homology
Function and homology information


catalase-peroxidase / catalase activity / hydrogen peroxide catabolic process / cellular response to hydrogen peroxide / heme binding / metal ion binding / cytosol
Similarity search - Function
Catalase-peroxidase haem / Peroxidases heam-ligand binding site / Peroxidase, active site / Peroxidases active site signature. / Plant heme peroxidase family profile. / Haem peroxidase / Peroxidase / Peroxidases proximal heme-ligand signature. / Haem peroxidase superfamily
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Catalase-peroxidase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.22 Å
AuthorsDuan, R. / Li, J. / Nathan, B. / Yang, X. / Liu, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM152982 United States
CitationJournal: Nat Commun / Year: 2026
Title: Single-atom substitution redirects KatG reactivity from cofactor biogenesis to stereoselective sulfoxidation.
Authors: Ran Duan / Jiasong Li / Wendell P Griffith / Yang Xu / Nathan D Burrows / Anthony P Green / Aimin Liu /
Abstract: Protein-derived cofactors rely on precisely positioned heteroatoms to direct redox chemistry, yet isolating their individual contributions remains challenging. The indole N-H of tryptophan plays a ...Protein-derived cofactors rely on precisely positioned heteroatoms to direct redox chemistry, yet isolating their individual contributions remains challenging. The indole N-H of tryptophan plays a central yet elusive role in biogenesis and function of the Met-Tyr-Trp (MYW) cofactor in catalase-peroxidase (KatG). Here, we use genetic code expansion to replace cofactor-forming Trp105 with thiotryptophan (S-Trp), enabling a single-heteroatom (N → S) substitution. Instead of forming the MYW crosslink, KatG bearing S-Trp105 undergoes site-specific monooxygenation to yield a chiral sulfoxide. HPLC-MS, circular dichroism, and FT-IR spectroscopy identify selective oxygen insertion at the sulfur, establishing enantioselective formation of an (S)-configured sulfoxide. A 2.22 Å cryo-EM structure visualizes the oxidized S-Trp105, revealing the S = O moiety orienting toward the iron and confirming the absence of crosslinking. The S-atom oxygenation is heme-dependent and proceeds via a two-electron oxygen-atom transfer, contrasting with the radical-mediated one-electron chemistry of native tryptophan. This redirection suppresses catalase activity by perturbing cofactor formation. These results show that a single-atom substitution reroutes the distal heme site from radical crosslinking to stereoselective sulfoxidation, uncovering a monooxygenase-like capability within KatG. This work highlights using noncanonical amino acids to achieve atomic-level control over reaction pathways and to interrogate cofactor biogenesis with unprecedented precision.
History
DepositionApr 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 22, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Catalase-peroxidase
B: Catalase-peroxidase
C: Catalase-peroxidase
D: Catalase-peroxidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)326,8898
Polymers324,4234
Non-polymers2,4664
Water36020
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
Catalase-peroxidase / CP / Peroxidase/catalase


Mass: 81105.672 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: katG, ECDH10B_4131 / Production host: Escherichia coli (E. coli) / References: UniProt: B1XBA8, catalase-peroxidase
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 20 / Source method: isolated from a natural source / Formula: H2O
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: EcKatG S-Trp105 / Type: COMPLEX
Details: KatG protein with 3-benzothienyl-L-alanine (S-Trp) genetically incorporated in place of W105
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.32 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 50 mM Tris-HCl, 50 mM NaCl at pH 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris(hydroxymethyl)aminomethane(HOCH2)3CNH21
250 mMSodium chlorideNaCl1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The KatG S-Trp105 was dissolved in the 50 mM Tris-HCl, 50 mM NaCl buffer at pH 8
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: 3 microliter of the sample was applied to each grid and blotted for 3 s

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.58 sec. / Electron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.5.3particle selectionBlob picking
4cryoSPARC4.5.3CTF correction
7PHENIX2.0rc1_5599model fitting
9PHENIX2.0rc1_5599model refinement
10cryoSPARC4.5.3initial Euler assignment
11cryoSPARC4.5.3final Euler assignment
12cryoSPARC4.5.3classification
13cryoSPARC4.5.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7762366 / Details: Particles were selected from blob picking.
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 884126 / Algorithm: FOURIER SPACE / Num. of class averages: 43 / Symmetry type: POINT
Atomic model buildingB value: 7.92 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7JZ6

7jz6


Pdb chain-ID: A / Accession code: 7JZ6 / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320868
ELECTRON MICROSCOPYf_angle_d0.5928384
ELECTRON MICROSCOPYf_dihedral_angle_d6.6492869
ELECTRON MICROSCOPYf_chiral_restr0.0422988
ELECTRON MICROSCOPYf_plane_restr0.0043720

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