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Oligomeric structures of metabolic protein assemblies - Structure-function analysis of acetyl-CoA carboxylase and urease

Anton, Leonie. Oligomeric structures of metabolic protein assemblies - Structure-function analysis of acetyl-CoA carboxylase and urease. 2020, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

Oligomeric protein assemblies play a pivotal role in metabolism. The advantages of such complexes are increased stability, protection from degradation and additional options for regulation through allostery. The two enzymes discussed in this thesis utilize oligomerization as a central mechanism, to shield from damaging conditions and control their activity.
Urease is a nickel-metalloenzyme with varying assembly structures expressed in all branches of life except animals and is an essential part of the nitrogen cycle. It catalyzes the breakdown of urea into ammonia, which is used as a nitrogen source. Oligomerization of urease leads to the local increase of ammonia in a concentrated area and contributes to urease stability in extreme environments. Ureases have a huge impact on agricultural practices, but their function as virulence factors in pathogens also makes them important drug targets.
Acetyl-CoA Carboxylase (ACC) catalyzes the first and rate limiting step in the production of fatty acids. Acetyl-CoA is carboxylated by ACC in two consecutive and distinct reactions, forming malonyl-CoA. Fatty acid synthase (FAS) uses malonyl-CoA as a precursor for the formation of fatty acids. Eukaryotic ACCs are multienzymes, which contain all the catalytic domains in a single polypeptide chain. Human ACC is a dimer and oligomerizes into a filament in its most active state. Upregulation of ACC activity has been linked to cancer, metabolic syndrome and viral infections.
The aim of this thesis is the investigation of the mechanisms leading to oligomerization of these essential enzymes by using cryo-electron microscopy (cryo-EM).
In chapter two, the nickel –metalloenzyme urease of Yersinia enterocolitica is revealed to form a tetramer-of-trimers. A similar assembly has only been observed for the pathogen Helicobacter pylori, where it is described as a crucial factor in survival of acidic environments. Including higher order aberration correction in data processing of cryoEM movies, a reconstruction of Y. enterocolitica urease at 1.98 Å resolution was obtained. The structural data revealed an oligomerization loop and a specific alpha-helix as central elements involved in the oligomerization. At better than 2.0 Å resolution, so far only reached by single-particle analysis for four others proteins, salt bridges, alternate conformations and protonation are visualized for the entire protein, and in particular the active site. The two nickel ions in the active site are at a distance of only 3.2 Å, much closer than in other urease active sites described so far by X-ray crystallography. Frame-wise analysis of cryoEM movies indicates the onset of radiation-damage in the environment of the Ni2+ ions during electron-beam exposure as the cause of a distorted overall bimetal center structure.
The mechanism of allosteric regulation of ACC is discussed in chapter three, uncovering the mechanism behind citrate-induced filament formation. Prior and newly collected cryoEM data of ACC filaments were processed using advanced algorithms. Corrections for higher order aberrations resulted in a 3.84 Å resolution reconstruction, which supported identification of a potential citrate interaction region. Site specific point mutants of residues in the suspected citrate binding pocket showed decreased activity in in vitro activity assays. Citrate acts on a pocket of positively charged residues in the central domain of the multienzyme. This central domain region has no catalytic properties, but was observed to function as a hinge where large conformational changes occur. Binding of citrate influences the conformational ensemble of the central domain region, creating an interface for other dimers of ACC to dock and form a filament. This filament locks ACC in an active conformation and is the most active state of the protein.
The findings of this thesis show how different proteins employ oligomerization to preserve or augment their function. Ureases grant protection from acidic environments through their assembly while citrate-induced filament formation increases ACC activity.
Advisors:Maier, Timm and Basler, Marek
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Maier)
UniBasel Contributors:Anton, Leonie and Maier, Timm and Basler, Marek
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13772
Thesis status:Complete
Number of Pages:122
Language:English
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss137721
edoc DOI:
Last Modified:15 Feb 2021 14:37
Deposited On:27 Jan 2021 15:29

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