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- PDB-6i00: Cryo-EM informed directed evolution of Nitrilase 4 leads to a cha... -

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Basic information

Entry
Database: PDB / ID: 6i00
TitleCryo-EM informed directed evolution of Nitrilase 4 leads to a change in quaternary structure.
ComponentsBifunctional nitrilase/nitrile hydratase NIT4
KeywordsHYDROLASE / NITRILASE / Cyanide detoxification / Beta-cyano-L-alanine hydrolase / Helical filament
Function / homology
Function and homology information


cyanoalanine nitrilase / 3-cyanoalanine hydratase / cyanoalanine nitrilase activity / 3-cyanoalanine hydratase activity / indole-3-acetonitrile nitrilase activity / cyanide metabolic process / nitrilase / nitrilase activity / detoxification of nitrogen compound / nitrile hydratase activity ...cyanoalanine nitrilase / 3-cyanoalanine hydratase / cyanoalanine nitrilase activity / 3-cyanoalanine hydratase activity / indole-3-acetonitrile nitrilase activity / cyanide metabolic process / nitrilase / nitrilase activity / detoxification of nitrogen compound / nitrile hydratase activity / plasma membrane / cytosol
Similarity search - Function
Nitrilases / cyanide hydratase active site signature. / Nitrilase/Cyanide hydratase / Nitrilases / cyanide hydratase signature 1. / Nitrilase/cyanide hydratase, conserved site / Carbon-nitrogen hydrolase domain profile. / Carbon-nitrogen hydrolase superfamily / Carbon-nitrogen hydrolase / Carbon-nitrogen hydrolase
Similarity search - Domain/homology
Bifunctional nitrilase/nitrile hydratase NIT4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMulelu, A.E. / Woodward, J.D.
Funding support South Africa, United Kingdom, 2items
OrganizationGrant numberCountry
National Research Foundation in South AfricaResearch Career Advancement Fellowship South Africa
Global Challenges Research FundStart: Synchrotron Techniques for African Research and Technology, ST/R002754/1 United Kingdom
CitationJournal: Commun Biol / Year: 2019
Title: Cryo-EM and directed evolution reveal how nitrilase specificity is influenced by its quaternary structure.
Authors: Andani E Mulelu / Angela M Kirykowicz / Jeremy D Woodward /
Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced ...Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from . We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.
History
DepositionOct 24, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
J: Bifunctional nitrilase/nitrile hydratase NIT4
I: Bifunctional nitrilase/nitrile hydratase NIT4
H: Bifunctional nitrilase/nitrile hydratase NIT4
G: Bifunctional nitrilase/nitrile hydratase NIT4
F: Bifunctional nitrilase/nitrile hydratase NIT4
E: Bifunctional nitrilase/nitrile hydratase NIT4
D: Bifunctional nitrilase/nitrile hydratase NIT4
C: Bifunctional nitrilase/nitrile hydratase NIT4
B: Bifunctional nitrilase/nitrile hydratase NIT4
A: Bifunctional nitrilase/nitrile hydratase NIT4
L: Bifunctional nitrilase/nitrile hydratase NIT4
K: Bifunctional nitrilase/nitrile hydratase NIT4


Theoretical massNumber of molelcules
Total (without water)477,19312
Polymers477,19312
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area56940 Å2
ΔGint-291 kcal/mol
Surface area124920 Å2
MethodPISA

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Components

#1: Protein
Bifunctional nitrilase/nitrile hydratase NIT4 / Cyanoalanine nitrilase / Nitrilase 4


Mass: 39766.113 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: NIT4, At5g22300, MWD9.8
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P46011, nitrilase, cyanoalanine nitrilase, 3-cyanoalanine hydratase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Active helical nitrilase complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMSodium chlorideNaCl1
250 mMTrisTris-Hcl1
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 293 K
Details: The sample (2.5 ul) was applied to the grid and incubated for 30 seconds at 50% humidity before blotting and plunging.

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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: 750 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 45.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1266
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 45

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Processing

EM software
IDNameVersionCategoryDetails
2EPU1.9.1image acquisition
4Gctf1.06CTF correction
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classificationRELION helical processing
12RELION23D reconstruction
13PHENIX1.14model refinementReal-space refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -72.98 ° / Axial rise/subunit: 17.48 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 392263
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 133106 / Num. of class averages: 67 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Ab initio fitting was performed using Buccaneer, cleaned up with Coot and Phenix Real-Space Refine.

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