During germination plants convert stored fatty acids into sucrose via β-oxidation, the glyoxylate cycle and gluconeogenesis, in order to supply energy prior to the
commencement of photosynthesis. One such fatty acid, acetyl-CoA, is produced both from β-oxidation and externally supplied acetate. Within the glyoxysome, externally supplied acetate is converted to acetyl-CoA by AAE7/ACN1, prior to entering the glyoxylate cycle. Structural analysis of AAE7/ACN1 has been conducted within this thesis to determine if AAE7/ACN1 is able to undergo a conformational change. Experimental and theoretical structural analysis has shown that AAE7/ACN1 is a dimeric protein in solution, although experimental analysis using solution scattering was unable to detect a conformational change in the presence and absence of a substrate analogue.
Previous research shows that Arabidopsis seedlings are able to take up and
metabolise both acetate and butyrate from the growth medium and shows that
aae7/acn1 mutant growth is inhibited as a result of cytosolic acidification. The
dimeric structure of AAE7/ACN1, identified within this thesis, results in a larger
concentration of active sites within a localised area. This may result in the speedy
detoxification of acetate and butyrate.
Other work in this thesis focuses on the role of aconitase within the glyoxylate and TCA cycles. Three isoforms of aconitase have previously been identified in the dicotyledonous plant Arabidopsis thaliana, namely ACOI, ACO2 and ACO3. A
cytosolic location has been suggested for ACO1, whilst ACO2 and ACO3 have been identified within the mitochondria. The cytosolic isoform of aconitase is believed to be involved in the glyoxylate cycle, where it converts citrate to isocitrate, though the specific role each isoform plays in metabolism during germination is unclear. A metabolic approach has been undertaken within this thesis to determine the role each isoform plays in seedling establishment. A large reduction in aconitase activity, an increase in citrate levels and a delay in germination were observed in aco3 mutants. These results suggest that ACO3 makes the largest contribution to citrate metabolism in germinating seedlings. This may mean that during seedling establishment the glyoxylate cycle is not as important as previously thought.
Active aconitase contains a [4Fe-4S] cluster. Under conditions of iron deficiency, the [4Fe-4S] cluster of aconitase is converted to a [3Fe-4S] cluster, which causes
mammalian aconitase to lose its enzymatic function and become an RNA binding
protein. A structural approach has been used within this thesis to attempt to
characterise the Arabidopsis aconitase isoforms. ACOI and ACO2 have been overexpressed in the eukaryotic organism Pichia pastoris. A novel method of overexpressing active aconitase without the need for reactivation has also been identified and implemented to show that both ACO1 and ACO2 are able to function as aconitase, confirming previously published work that used mutagenic analysis.