Synthetic vesicles for metabolic energy conservation

Tjeerd Pols

Research output: ThesisThesis fully internal (DIV)

57 Downloads (Pure)

Abstract

Cells can generate and conserve metabolic energy with complex pathways, like oxidative phosphorylation, or simpler pathways, like deamination of amino acids, oxidation of carboxylic acids or using light. This thesis focusses on deamination of arginine to create adenosine triphosphate (ATP) inside synthetic vesicles, which is carried out by the arginine deiminase pathway in cells. This pathway consists of three cytosolic proteins (arginine deiminase, ornithine transcarbamoylase and carbamate kinase) and one arginine/ornithine antiporter.
First, these proteins were purified from Lactococcus lactis and characterized, to determine their kinetic constants and the effect of varying conditions on their activity. The enzymes perform their reactions as expected and work relatively well in the tested conditions. The antiporter is relatively slow and possibly the rate-determining reaction of the pathway.
Next, the pathway was reconstituted in synthetic vesicles, shifting the focus from the individual proteins to measuring properties of the entire pathway. These properties were measured by studying internal pH, metabolic energy levels and concentrations of arginine, ornithine and citrulline. Without an ATP-consuming process, arginine is found to be hydrolyzed into citrulline, which is a wasteful side reaction that hinders the conservation of metabolic energy.
Finally, the ATP-driven transporter OpuA was added to the vesicles, which imports the compatible solute glycine betaine for volume regulation in osmotically stressed vesicles. The addition of OpuA does not only help as an ATP-consuming process, but the presence of glycine betaine protects the proteins from osmotic stress. This leads to higher metabolic energy levels and thus metabolic energy conservation.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • University of Groningen
Supervisors/Advisors
  • Poolman, Berend, Supervisor
  • Heinemann, Matthias, Supervisor
Award date20-Nov-2020
Place of Publication[Groningen]
Publisher
DOIs
Publication statusPublished - 2020

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