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- PDB-9pxt: Cryo-EM structure of NapA, the periplasmic nitrate reductase from... -

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Basic information

Entry
Database: PDB / ID: 9pxt
TitleCryo-EM structure of NapA, the periplasmic nitrate reductase from Campylobacter jejuni
ComponentsPeriplasmic nitrate reductase
KeywordsOXIDOREDUCTASE / Periplasmic nitrate reductase / molybdenum enzymes / cryo-EM / MEMBRANE PROTEIN / METAL BINDING PROTEIN
Function / homology
Function and homology information


nitrate reductase (cytochrome) / nitrate reductase (cytochrome) activity / nitrate reductase complex / Mo-molybdopterin cofactor biosynthetic process / molybdenum ion binding / molybdopterin cofactor binding / cellular respiration / nitrate assimilation / 4 iron, 4 sulfur cluster binding / periplasmic space ...nitrate reductase (cytochrome) / nitrate reductase (cytochrome) activity / nitrate reductase complex / Mo-molybdopterin cofactor biosynthetic process / molybdenum ion binding / molybdopterin cofactor binding / cellular respiration / nitrate assimilation / 4 iron, 4 sulfur cluster binding / periplasmic space / electron transfer activity / iron ion binding / membrane
Similarity search - Function
Periplasmic nitrate reductase, large subunit / Nitrate reductase NapA-like, molybdopterin-binding domain / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin dinucleotide-binding domain / Molydopterin dinucleotide binding domain / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : ...Periplasmic nitrate reductase, large subunit / Nitrate reductase NapA-like, molybdopterin-binding domain / Molybdopterin oxidoreductase, molybdopterin cofactor binding site / Prokaryotic molybdopterin oxidoreductases signature 1. / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin oxidoreductase Fe4S4 domain / Molybdopterin dinucleotide-binding domain / Molydopterin dinucleotide binding domain / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : / Aspartate decarboxylase-like domain superfamily / Molybdopterin oxidoreductase, 4Fe-4S domain / Prokaryotic molybdopterin oxidoreductases 4Fe-4S domain profile. / Molybdopterin oxidoreductase / Molybdopterin oxidoreductase
Similarity search - Domain/homology
Chem-MGD / MOLYBDENUM ATOM / IRON/SULFUR CLUSTER / Periplasmic nitrate reductase
Similarity search - Component
Biological speciesCampylobacter jejuni (Campylobacter)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsThach, T. / Subramanian, R.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Biol Chem / Year: 2025
Title: Structure and substrate promiscuity of Campylobacter jejuni periplasmic nitrate reductase (Nap) and phylogenetic analysis of Nap homologs.
Authors: Nitai C Giri / Trung Thach / KanagaVijayan Dhanabalan / Mintare Cesiunaite / Manohar Radhakrishnan / Lahiru Wedasingha / Nicholas Manicke / Michael Wells / Maciej Szaleniec / Ramaswamy ...Authors: Nitai C Giri / Trung Thach / KanagaVijayan Dhanabalan / Mintare Cesiunaite / Manohar Radhakrishnan / Lahiru Wedasingha / Nicholas Manicke / Michael Wells / Maciej Szaleniec / Ramaswamy Subramanian / Partha Basu /
Abstract: Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger ...Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger compared to other structurally characterized NapA. Herein, we present the cryo-EM structure of CjNapA, the first of its kind from any ε-proteobacteria, revealing three lysine-rich insertions that could affect the substrate channel, potentially enhancing the affinity towards nitrate and other anionic substrates. Here, we report that wild-type CjNapA and NapA-C176D variants can reduce chlorate, perchlorate, and nitrate. However, the perchlorate and chlorate reductions by the CjNapA C176D variant are considerably slower, even though the perchlorate reductase has an Asp coordination to Mo. Molecular Dynamics (MD) simulations were performed to investigate the impact of the C176D mutation on substrate affinity and protein flexibility. Structural and kinetic comparisons with perchlorate reductase support evolutionary tuning for a desired function. Finally, structural comparisons with other structurally characterized NapAs also suggest the role of proximal pterin in CjNapA in electron transfer to the Mo center.
History
DepositionAug 6, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 17, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 17, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Periplasmic nitrate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)107,0225
Polymers105,0931
Non-polymers1,9294
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 Periplasmic nitrate reductase


Mass: 105092.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Campylobacter jejuni (Campylobacter) / Gene: napA, Cj0780 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9PPD9, nitrate reductase (cytochrome)
#2: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-MO / MOLYBDENUM ATOM


Mass: 95.940 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mo / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-MGD / 2-AMINO-5,6-DIMERCAPTO-7-METHYL-3,7,8A,9-TETRAHYDRO-8-OXA-1,3,9,10-TETRAAZA-ANTHRACEN-4-ONE GUANOSINE DINUCLEOTIDE / MOLYBDOPTERIN GUANOSINE DINUCLEOTIDE


Mass: 740.557 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C20H26N10O13P2S2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Structure of NapA:Moco[4Fe4S] / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.084 MDa / Experimental value: YES
Source (natural)Organism: Campylobacter jejuni (Campylobacter)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 25 mM Hepes 7.0, 100 mM NaCl, 0.5 mM TCEP, 2% glycerol
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2100 mMSodium ChlorideNaCl1
30.5 mMTris(2-carboxyethyl)phosphineTCEP1
42 %GlycerolGlycerol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: vitrification

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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 defocus max: 2000 nm / Nominal defocus min: 700 nm / Cs: 0.1 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 56.8 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 60
EM imaging opticsEnergyfilter name: GIF Bioquantum

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIXdev_5533model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 200982
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131448 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Details: real refinement was done using Phenix
Atomic model buildingChain residue range: 1-924 / Details: The initial model consisted of monomer / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 34.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00437144
ELECTRON MICROSCOPYf_angle_d0.60759666
ELECTRON MICROSCOPYf_chiral_restr0.04531002
ELECTRON MICROSCOPYf_plane_restr0.00491221
ELECTRON MICROSCOPYf_dihedral_angle_d16.18852706

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