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- EMDB-19897: CryoEM Structure of Phenylalanine Ammonia Lyase from Planctomyces... -

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Basic information

Entry
Database: EMDB / ID: EMD-19897
TitleCryoEM Structure of Phenylalanine Ammonia Lyase from Planctomyces brasiliencis
Map data
Sample
  • Complex: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-2-phenylethyl]phosphonic acid
    • Protein or peptide: Histidine ammonia-lyase
  • Ligand: [(1R)-1-amino-2-phenylethyl]phosphonic acid
KeywordsPhenylalanine catabolism Lyase / LYASE
Function / homologyhistidine ammonia-lyase / histidine ammonia-lyase activity / Aromatic amino acid lyase / Aromatic amino acid lyase / Fumarase/histidase, N-terminal / L-Aspartase-like / secondary metabolite biosynthetic process / Histidine ammonia-lyase
Function and homology information
Biological speciesRubinisphaera brasiliensis DSM 5305 (bacteria) / Rubinisphaera brasiliensis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.17 Å
AuthorsDuhoo Y / Buslov I / Desmons S
Funding support Switzerland, 1 items
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, 2024-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19897.png

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Map

FileDownload / File: emd_19897.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 288 pix.
= 232.33 Å		0.81 Å/pix.
x 288 pix.
= 232.33 Å		0.81 Å/pix.
x 288 pix.
= 232.33 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8067 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.97550786 - 1.4629864
Average (Standard dev.)0.0005135142 (±0.05422818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 232.32959 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19897_msk_1.map
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Half map: #1

Fileemd_19897_half_map_1.map
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Half map: #2

Fileemd_19897_half_map_2.map
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Sample components

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Entire : Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-...

EntireName: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-2-phenylethyl]phosphonic acid
Components
  • Complex: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-2-phenylethyl]phosphonic acid
    • Protein or peptide: Histidine ammonia-lyase
  • Ligand: [(1R)-1-amino-2-phenylethyl]phosphonic acid

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Supramolecule #1: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-...

SupramoleculeName: Phenylalanine ammonia-lyase protein in complex with (1R)-1-amino-2-phenylethyl]phosphonic acid
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Rubinisphaera brasiliensis DSM 5305 (bacteria)

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Macromolecule #1: Histidine ammonia-lyase

MacromoleculeName: Histidine ammonia-lyase / type: protein_or_peptide / ID: 1 / Details: MDO: non standard Amino acid ALA-SER-GLY / Number of copies: 4 / Enantiomer: LEVO / EC number: histidine ammonia-lyase
Source (natural)Organism: Rubinisphaera brasiliensis (bacteria)
Molecular weightTheoretical: 62.710332 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLASSPSGHT NPVLSGAPLS INVVADIGRQ RLIPSLTDDE QVLNRVHACR DVVQKAVRNN ERIYGITTGF GGMSDIPIPP QHVAQTQDN LLAFLSTSTG ASLDPRHVRA AMALRANVLL QGRSGVRLEL IERLVEFLRQ DAIPVVCDLG SIG(MDO)DLV PL GVIARSIIGH ...String:
MLASSPSGHT NPVLSGAPLS INVVADIGRQ RLIPSLTDDE QVLNRVHACR DVVQKAVRNN ERIYGITTGF GGMSDIPIPP QHVAQTQDN LLAFLSTSTG ASLDPRHVRA AMALRANVLL QGRSGVRLEL IERLVEFLRQ DAIPVVCDLG SIG(MDO)DLV PL GVIARSIIGH PSTTQVKYQG EQADSHDVLQ QLNYSALQLE AKEGLALVNG TSFSSAIAAN CVFESQRLLS LSLVLQSI M VRALGGHPEA FHPFVDENKP HPGQGWSAQM MRDLLSYSPN DSKRNGDLAQ DRYSLRCLAQ YFAPIVEGIA QISQSISTE MNAVSDNPLI DVDTGRFHQS GNFLGQYVAM SMDQLRRHLG LLAKHLDVQI AQLVAPAFNN GLPASLRGNS SRPFNMGLKG LQITGNSIM PLLTYLGNPL TEHFPTHAEE FNQNINGLSW GSANLAWRSV QLFQHYLSVA SIFAVQAIDL RAGLEADHCD G RELLGETA TELYETVYDL LERNCGQESP FLFNDDEQSL EVDLQMLNGD LAGAGRMHEA VSSVTDSFLA EFCESGGGLE VL FQGPGGS SGSGHHHHHH HHH

UniProtKB: Histidine ammonia-lyase

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Macromolecule #2: [(1R)-1-amino-2-phenylethyl]phosphonic acid

MacromoleculeName: [(1R)-1-amino-2-phenylethyl]phosphonic acid / type: ligand / ID: 2 / Number of copies: 4 / Formula: PPH
Molecular weightTheoretical: 201.16 Da
Chemical component information

ChemComp-PPH:
[(1R)-1-amino-2-phenylethyl]phosphonic acid

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8.8
Component:
ConcentrationFormulaName
300.0 mMNaClsodium Chloride
50.0 mMTrisTris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3171027
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.17 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 95257
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 1 / Avg.num./class: 95257
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6-f1


Chain - Chain ID: A / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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