| Our lab has been interested in studying the initial steps of the de novo pathway leading up to the synthesis of the pyrimidine ring (dihydroorotate, DHO). The first two steps of the pathway are responsible for assembling the backbone of the pyrimidine ring (carbamoyl aspartate, C-Asp) while the third involves ring closure to form DHO. First, glutamine-dependent carbamoyl phosphate synthetase (CPSase, consists of glutaminase and synthetase) catalyzes the synthesis of carbamoyl phosphate (CP) using two molecules of ATP, glutamine (Gin) and bicarbonate. Next, aspartate transcarbamoylase (ATCase) synthesizes C-Asp by the addition of aspartate (Asp). Finally, dihydroorotase (DHOase) brings about a condensation reaction to close the ring and form DHO. In eukaryotes, this entire reaction is carried out by a single multifunctional polypeptide chain. In Escherichia coli, on the other hand, each reaction is carried out by a separate protein.; My doctoral work has focused on characterizing these enzymes from the hyperthermophile, Aquifex aeolicus. The motivation to use this organism as the model is two-fold. First, sequence analysis revealed that, unlike any other organism, the CPSase from A. aeolicus is encoded by two separate genes, corresponding to the two homologous halves of the enzyme. These two subdomains of CPSase catalyze the two ATP-dependent partial reactions involved in CP synthesis. In E. coli and mammals, these two subdomains have been shown to be redundant when cloned individually. Therefore, we were interested in studying if the individual 'halves' of A. aeolicus were sufficient to catalyze the entire reaction. Second, analysis of pathways operating at high temperatures could reveal novel adaptations to circumvent its detrimental effects. For instance, CP is extremely thermolabile with a half-life of 2 seconds at 100°C, decaying to form cyanate. Therefore, it raises the question how a hyperthermophile like A. aeolicus manages to utilize CP efficiently before it is degraded. In addition, by analyzing all the reactions that lead up to the synthesis of DHO, we have obtained some insight into the coordination of the entire pathway through the interactions between individual enzymes with each other. |
