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- EMDB-17778: Engineered glycolyl-CoA carboxylase (G20R variant) with bound CoA -

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Basic information

Entry
Database: EMDB / ID: EMD-17778
TitleEngineered glycolyl-CoA carboxylase (G20R variant) with bound CoA
Map datamain map
Sample
  • Complex: glycolyl-CoA carboxylase with bound CoA
    • Protein or peptide: Propionyl-CoA carboxylase beta chain
    • Protein or peptide: Propionyl-CoA carboxylase alpha subunit
  • Ligand: COENZYME A
  • Ligand: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL
  • Ligand: water
Keywordsglycolyl-CoA carboxylase / LIGASE
Function / homology
Function and homology information


propionyl-CoA carboxylase / propionyl-CoA carboxylase activity / lipid catabolic process / ATP binding / metal ion binding
Similarity search - Function
Propionyl-coenzyme A carboxylase, BT domain / Propionyl-coenzyme A carboxylase BT domain / Acetyl-coenzyme A carboxyltransferase, C-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase C-terminal domain profile. / Acetyl-coenzyme A carboxyltransferase, N-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase N-terminal domain profile. / Acetyl-CoA carboxylase / Carboxyl transferase domain / Biotin-binding site / Biotin-requiring enzymes attachment site. ...Propionyl-coenzyme A carboxylase, BT domain / Propionyl-coenzyme A carboxylase BT domain / Acetyl-coenzyme A carboxyltransferase, C-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase C-terminal domain profile. / Acetyl-coenzyme A carboxyltransferase, N-terminal / Acetyl-coenzyme A (CoA) carboxyltransferase N-terminal domain profile. / Acetyl-CoA carboxylase / Carboxyl transferase domain / Biotin-binding site / Biotin-requiring enzymes attachment site. / Biotin carboxylase-like, N-terminal domain / Biotin carboxylase, C-terminal / Biotin carboxylation domain / Biotin carboxylase, N-terminal domain / Biotin carboxylase C-terminal domain / Biotin carboxylation domain profile. / Biotin carboxylase C-terminal domain / Carbamoyl-phosphate synthase subdomain signature 1. / Carbamoyl-phosphate synthetase large subunit-like, ATP-binding domain / Carbamoyl-phosphate synthase L chain, ATP binding domain / Biotin-requiring enzyme / Biotinyl/lipoyl domain profile. / Biotin/lipoyl attachment / Rudiment single hybrid motif / Single hybrid motif / Pre-ATP-grasp domain superfamily / ATP-grasp fold / ATP-grasp fold profile. / ClpP/crotonase-like domain superfamily / Carbamoyl-phosphate synthase subdomain signature 2.
Similarity search - Domain/homology
Propionyl-CoA carboxylase beta chain / propionyl-CoA carboxylase
Similarity search - Component
Biological speciesMethylorubrum extorquens AM1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsZarzycki J / Marchal DG / Schulz L / Prinz S / Erb TJ
Funding supportEuropean Union, Germany, 3 items
OrganizationGrant numberCountry
European Commission862087European Union
Max Planck Society Germany
Joachim Herz Stiftung Germany
CitationJournal: ACS Synth Biol / Year: 2023
Title: Machine Learning-Supported Enzyme Engineering toward Improved CO-Fixation of Glycolyl-CoA Carboxylase.
Authors: Daniel G Marchal / Luca Schulz / Ingmar Schuster / Jelena Ivanovska / Nicole Paczia / Simone Prinz / Jan Zarzycki / Tobias J Erb /
Abstract: Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve ...Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO fixation. To further improve upon GCC M5, we developed a machine learning-supported workflow that reduces screening efforts for identifying improved enzymes. Using this workflow, we present two novel GCC variants with 2-fold increased carboxylation rate and 60% reduced energy demand, respectively, which are able to address kinetic and thermodynamic limitations of the TaCo pathway. Our work highlights the potential of combining machine learning and directed evolution strategies to reduce screening efforts in enzyme engineering.
History
DepositionJun 30, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateDec 27, 2023-
Current statusDec 27, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17778.png

Downloads & links

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Map

FileDownload / File: emd_17778.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Voxel sizeX=Y=Z: 0.837 Å
Density
Contour LevelBy AUTHOR: 0.09
Minimum - Maximum-0.7776795 - 1.4050127
Average (Standard dev.)0.00017948337 (±0.032018308)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 418.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_17778_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_17778_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : glycolyl-CoA carboxylase with bound CoA

EntireName: glycolyl-CoA carboxylase with bound CoA
Components
  • Complex: glycolyl-CoA carboxylase with bound CoA
    • Protein or peptide: Propionyl-CoA carboxylase beta chain
    • Protein or peptide: Propionyl-CoA carboxylase alpha subunit
  • Ligand: COENZYME A
  • Ligand: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL
  • Ligand: water

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Supramolecule #1: glycolyl-CoA carboxylase with bound CoA

SupramoleculeName: glycolyl-CoA carboxylase with bound CoA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Methylorubrum extorquens AM1 (bacteria)

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Macromolecule #1: Propionyl-CoA carboxylase beta chain

MacromoleculeName: Propionyl-CoA carboxylase beta chain / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: propionyl-CoA carboxylase
Source (natural)Organism: Methylorubrum extorquens AM1 (bacteria)
Molecular weightTheoretical: 55.990953 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKDILEKLEE RRAQARLGGG EKRLEAQHKR GKLTARERIE LLLDHGSFEE FDMFVQHRST DFGMEKQKIP GDGVVTGWGT VNGRTVFLF SKDFTVFGGS NSEAHAAKIV KVQDMALKMR APIIGIFDAG GARIQEGVAA LGGHGEVFRR NVAASGVIPQ I SVIMGPCA ...String:
MKDILEKLEE RRAQARLGGG EKRLEAQHKR GKLTARERIE LLLDHGSFEE FDMFVQHRST DFGMEKQKIP GDGVVTGWGT VNGRTVFLF SKDFTVFGGS NSEAHAAKIV KVQDMALKMR APIIGIFDAG GARIQEGVAA LGGHGEVFRR NVAASGVIPQ I SVIMGPCA GGDVYSPAMT DFIFMVRDTS YMFVTGPDVV KTVTNEVVTA EELGGAKVHT SKSSIADGSF ENDVEAILQI RR LLDFLPA NNIEGVPEIE SFDDVNRLDK SLDTLIPDNP NKPYDMGELI RRVVDEGDFF EIQAAYARNI ITGFGRVEGR TVG FVANQP LVLAGVLDSD ASRKAARFVR FCNAFSIPIV TFVDVPGFLP GTAQEYGGLI KHGAKLLFAY SQATVPLVTI ITRK AFGGA YIVMASKHVG ADLNYAWPTA QIAVMGAKGA VEIIFRAEIG DADKVAERTK EYEDRFLSPF VAAERGYIDE VIMPH STRK RIARALGMLR TKEMEQPRKK HDNIPL

UniProtKB: Propionyl-CoA carboxylase beta chain

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Macromolecule #2: Propionyl-CoA carboxylase alpha subunit

MacromoleculeName: Propionyl-CoA carboxylase alpha subunit / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO / EC number: propionyl-CoA carboxylase
Source (natural)Organism: Methylorubrum extorquens AM1 (bacteria)
Molecular weightTheoretical: 71.986961 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MFDKILIANR GEIACRIIKT AQKMGIKTVA VYSDADRDAV HVAMADEAVH IGPAPAAQSY LLIEKIIDAC KQTGAQAVHP GYGFLSERE SFPKALAEAG IVFIGPNPGA IAAMGDKIES KKAAAAAEVS TVPGFLGVIE SPEHAVTIAD EIGYPVMIKA S AGGGGKGM ...String:
MFDKILIANR GEIACRIIKT AQKMGIKTVA VYSDADRDAV HVAMADEAVH IGPAPAAQSY LLIEKIIDAC KQTGAQAVHP GYGFLSERE SFPKALAEAG IVFIGPNPGA IAAMGDKIES KKAAAAAEVS TVPGFLGVIE SPEHAVTIAD EIGYPVMIKA S AGGGGKGM RIAESADEVA EGFARAKSEA SSSFGDDRVF VEKFITDPRH IEIQVIGDKH GNVIYLGERE CSIQRRNQKV IE EAPSPLL DEETRRKMGE QAVALAKAVN YDSAGTVEFV AGQDKSFYFL EMNTRLQVEH PVTEMITGLD LVELMIRVAA GEK LPLSQD QVKLDGWAVE SRVYAEDPTR NFLPSIGRLT TYQPPEEGPL GGAIVRNDTG VEEGGEIAIH YDPMIAKLVT WAPT RLEAI EAQATALDAF AIEGIRHNIP FLATLMAHPR WRDGRLSTGF IKEEFPEGFI APEPEGPVAH RLAAVAAAID HKLNI RKRG ISGQMRDPSL LTFQRERVVV LSGQRFNVTV DPDGDDLLVT FDDGTTAPVR SAWRPGAPVW SGTVGDQSVA IQVRPL LNG VFLQHAGAAA EARVFTRREA ELADLMPVKE NAGSGKQLLC PMPGLVKQIM VSEGQEVKNG EPLAIVEAMK MENVLRA ER DGTISKIAAK EGDSLAVDAV ILEFA

UniProtKB: propionyl-CoA carboxylase

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Macromolecule #3: COENZYME A

MacromoleculeName: COENZYME A / type: ligand / ID: 3 / Number of copies: 6 / Formula: COA
Molecular weightTheoretical: 767.534 Da
Chemical component information

ChemComp-COA:
COENZYME A / Coenzyme A

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Macromolecule #4: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL

MacromoleculeName: 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL
type: ligand / ID: 4 / Number of copies: 6 / Formula: BTI
Molecular weightTheoretical: 228.311 Da
Chemical component information

ChemComp-BTI:
5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 866 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

BufferpH: 7.8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ybq

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 113824
FSC plot (resolution estimation)

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