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- PDB-8pn8: Engineered glycolyl-CoA carboxylase (L100N variant) with bound CoA -

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Basic information

Entry
Database: PDB / ID: 8pn8
TitleEngineered glycolyl-CoA carboxylase (L100N variant) with bound CoA
Components
  • Propionyl-CoA carboxylase alpha subunit
  • Propionyl-CoA carboxylase beta chain
KeywordsLIGASE / glycolyl-CoA carboxylase
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
Chem-BTI / COENZYME A / Propionyl-CoA carboxylase beta chain / propionyl-CoA carboxylase
Similarity search - Component
Biological speciesMethylorubrum extorquens AM1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsZarzycki, J. / Marchal, D.G. / Schulz, L. / Prinz, S. / Erb, T.J.
Funding supportEuropean Union, Germany, 3items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 27, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Propionyl-CoA carboxylase beta chain
B: Propionyl-CoA carboxylase beta chain
C: Propionyl-CoA carboxylase beta chain
D: Propionyl-CoA carboxylase beta chain
E: Propionyl-CoA carboxylase beta chain
F: Propionyl-CoA carboxylase beta chain
G: Propionyl-CoA carboxylase alpha subunit
H: Propionyl-CoA carboxylase alpha subunit
I: Propionyl-CoA carboxylase alpha subunit
J: Propionyl-CoA carboxylase alpha subunit
K: Propionyl-CoA carboxylase alpha subunit
L: Propionyl-CoA carboxylase alpha subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)773,84324
Polymers767,86712
Non-polymers5,97512
Water15,601866
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area76780 Å2
ΔGint-329 kcal/mol
Surface area151230 Å2

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Components

#1: Protein
Propionyl-CoA carboxylase beta chain


Mass: 55990.953 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methylorubrum extorquens AM1 (bacteria)
Gene: pccB, MexAM1_META1p0172 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5AP75, propionyl-CoA carboxylase
#2: Protein
Propionyl-CoA carboxylase alpha subunit


Mass: 71986.961 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Methylorubrum extorquens AM1 (bacteria)
Gene: pccA, MexAM1_META1p3203 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: C5AWU5, propionyl-CoA carboxylase
#3: Chemical
ChemComp-COA / COENZYME A / Coenzyme A


Mass: 767.534 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C21H36N7O16P3S / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-BTI / 5-(HEXAHYDRO-2-OXO-1H-THIENO[3,4-D]IMIDAZOL-6-YL)PENTANAL


Mass: 228.311 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N2O2S
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 866 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: glycolyl-CoA carboxylase with bound CoA / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Methylorubrum extorquens AM1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113824 / Symmetry type: POINT

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