AbstractMetabolism encompasses the biochemical basis of life, but even in well studied model organisms our knowledge of metabolism is incomplete (Downs, 2006). Sugars represent a major source of carbon supporting heterotrophic growth in Archaea and are oxidized via a conserved set of central metabolic pathways, including modified versions of the Entner-Doudoroff (ED) pathway. Sulfolobus solfataricus is a hyperthermophilic archaeon from the kingdom crenarchaeota. It is a strict aerobe that grows optimally at 80-85C, pH 2-4, and utilises numerous carbon sources including the four most-commonly occurring sugars in nature, D-glucose, D-galactose, D-xylose and L-arabinose. In S. solfataricus, glucose and galactose are metabolised through the non-phosphorylative ED pathway in which a single set of enzymes are utilised for the catabolism of both sugars, leading to the proposal of a metabolically promiscuous pathway. The first section of this pathway includes three enzymes, glucose dehydrogenase (GDH), gluconate dehydratase (GD) and 2-keto-3-deoxy-gluconate (KDG) aldolase, two of which, GDH and KDG aldolase, are found to have activity with pentose as well as hexose sugars, although the third enzyme, GD, is hexose specific. KDG-aldolase cleaves hexose substrates into pyruvate and glyceraldehyde, and pentoses into pyruvate and the C-2 compound glycoaldehyde. Glyceraldehyde is converted into a second molecule of pyruvate, but the fate of glycoaldehyde is currently unknown. In this thesis, the complete pathway for D-xylose and L-arabinose catabolism is elucidated. Through the detection of enzyme activities in the native organism and subsequent characterisation of recombinantly produced enzymes, the results presented show that a separate C5 dehydratase exists, completing the first part of a non-phosphorylative C5 pathway, and that the subsequent enzymes required for glycolaldehyde metabolism (glycolaldehyde oxidoreductase, glycolate oxidase and malate synthase) are present and active during growth on C5 sugars. In addition to the metabolic investigations, rational design and site-directed mutagenesis were used to explore the structural basis of substrate promiscuity of glucose dehydrogenase; in doing so, the enzyme was successfully engineered to oxidise the sugar D-ribose while retaining its activity with its natural substrates. These same techniques have been used to explore the relationship between structure, stability and catalytic activity of the S. solfataricus malate synthase, and the data have been fitted to a quantitative kinetic model that explores how temperature influences the activity of an enzyme.
|Date of Award||1 Jul 2010|
|Supervisor||Michael Danson (Supervisor) & David Hough (Supervisor)|
The Enzymology of Sugar Metabolism in Sulfolobus solfataricus
Nunn, C. (Author). 1 Jul 2010
Student thesis: Doctoral Thesis › PhD