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- PDB-9eq5: CryoEM Structure of Phenylalanine Ammonia Lyase from Planctomyces... -

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Basic information

Entry
Database: PDB / ID: 9eq5
TitleCryoEM Structure of Phenylalanine Ammonia Lyase from Planctomyces brasiliencis
ComponentsHistidine ammonia-lyase
KeywordsLYASE / Phenylalanine catabolism Lyase
Function / homology
Function and homology information


histidine ammonia-lyase / histidine ammonia-lyase activity / phenylalanine ammonia-lyase activity / secondary metabolite biosynthetic process
Similarity search - Function
Aromatic amino acid lyase / Aromatic amino acid lyase / Fumarase/histidase, N-terminal / L-Aspartase-like
Similarity search - Domain/homology
[(1R)-1-amino-2-phenylethyl]phosphonic acid / Histidine ammonia-lyase
Similarity search - Component
Biological speciesRubinisphaera brasiliensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.17 Å
AuthorsDuhoo, Y. / Buslov, I. / Desmons, S.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Angew Chem Int Ed Engl / Year: 2024
Title: Engineered Phenylalanine Ammonia-Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives.
Authors: Ivan Buslov / Sarah Desmons / Yoan Duhoo / Xile Hu /
Abstract: Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to ...Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron-deficient substrates is often poor. Here, we report the structure-based engineering of PAL from Planctomyces brasiliensis (PbPAL), enabling preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Through the elucidation of cryo-electron microscopy (cryo-EM) PbPAL structure and screening of the structure-based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron-deficient substrates. Our engineered PALs demonstrated exclusive α-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis.
History
DepositionMar 20, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Histidine ammonia-lyase
B: Histidine ammonia-lyase
C: Histidine ammonia-lyase
D: Histidine ammonia-lyase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)251,6468
Polymers250,8414
Non-polymers8054
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Histidine ammonia-lyase


Mass: 62710.332 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: MDO: non standard Amino acid ALA-SER-GLY / Source: (gene. exp.) Rubinisphaera brasiliensis (bacteria) / Gene: Plabr_3153 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: F0SIS6, histidine ammonia-lyase
#2: Chemical
ChemComp-PPH / [(1R)-1-amino-2-phenylethyl]phosphonic acid


Type: peptide-like / Mass: 201.160 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H12NO3P / Feature type: SUBJECT OF INVESTIGATION
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: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-2-phenylethyl]phosphonic acid
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rubinisphaera brasiliensis DSM 5305 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.8
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium ChlorideNaCl1
250 mMTrisTris1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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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 magnification: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3171027
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95257 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingAccession code: AF-F0SIS6-F1 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416080
ELECTRON MICROSCOPYf_angle_d0.64421836
ELECTRON MICROSCOPYf_dihedral_angle_d13.9545908
ELECTRON MICROSCOPYf_chiral_restr0.042496
ELECTRON MICROSCOPYf_plane_restr0.0052908

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