This project focuses on de novo pyrimidine biosynthesis in the hyperthermophilic organisms. Pyrococcus abyssi, an archaebacterium recently isolated from a submarine hydrothermal vent, has an optimum growth temperature of 96oC. Carbamoyl phosphate, one of the key metabolites in the pathway, is very unstable with a half life of 2 sec at this temperature and some of the intermediates involved in its synthesis are even less stable. Moreover, carbamoyl phosphate decomposes to cyanate, an indiscriminate and highly toxic carbamoylating agent. When Pyrococcus abyssi carbamoyl phosphate synthetase (CPSase) was cloned and expressed in E. coli, it was found to have a distinctly different structure and sequence compared to all known mesophilic CPSase molecules. In contrast, the recombinant aspartate transcarbamoylase (ATCase), the enzyme that catalyzes the next step in the de novo pyrimidine pathway appears to be quite similar to its mesophilic counterparts. This research is based on the hypothesis that carbamoyl phosphate is stabilized by channeling, a process that allows direct transfer of the intermediate from its site of synthesis on CPSase to ATCase where it is used to make carbamoyl aspartate. Partial channeling was found to appreciably reduce the steady state concentration of the intermediate, and thus its hydrolysis. However, the prerequisite physical association of CPSase and ATCase needed to sequester the intermediate could not be detected. Consequently, experiments are planned to quantitatively assess the factors that influence channeling and to look for physical and functional interactions between the enzymes. Possible mechanisms which protect these intermediates from thermal degradation are of special interest, not only because they represent an important aspect of the biology of these fascinating organisms, but also because they could have important biotechnological implications in the design of enzymes which function at elevated temperatures.
Pyrococcus abyssi, an archebacterium isolated from a deep-sea hydrothermal vent, flourishes at temperatures close to 100oC. Many metabolites are very unstable at these elevated temperatures and some breakdown to highly toxic compounds. Thus, mechanisms must have evolved in these organisms that protect the metabolic intermediates from thermal degradation. This research is based on the hypothesis that the intermediates are directly transferred or channeled between enzymes that catalyze sequential reactions in a metabolic pathway. Experiments are planned to determine whether channeling occurs and to detect the enzyme complexes responsible for sequestering the intermediates. These studies focus on an important aspect of the biology of these fascinating organisms, and moreover, have biotechnological implications for the design of enzymes which function at elevated temperatures.
