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Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum
M E Wales1, M G Mann-Dean, J R Wild
1Department of Biochemistry and Biophysics, Texas A&M University System, College Station 77843-2128.
Abstract:
The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.
Insights
Dictyostelium discoideum pyrimidine biosynthesis involves de novo and salvage pathways. During development, nucleotide pools drastically decrease, but energy charge is maintained, requiring cell-cell contact.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Pyrimidine biosynthesis is crucial for cellular function.
- Dictyostelium discoideum offers a model for studying nucleotide metabolism during development.
Purpose of the Study:
- To elucidate the de novo and salvage pyrimidine biosynthetic pathways in Dictyostelium discoideum.
- To investigate the changes in nucleotide metabolism during Dictyostelium discoideum development.
Main Methods:
- Enzyme activity assays for carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase.
- Analysis of molecular species and enzyme complex structures.
- Measurement of ribonucleotide pool levels, adenylate energy charge, and GTP/ATP ratios during starvation-induced development.
Main Results:
- Identified key enzymes in the arginine-independent de novo pyrimidine pathway, including carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase.
- Observed unstable molecular species suggesting potential multienzyme complexes.
- Demonstrated salvage utilization of uracil, uridine, and cytidine.
- Showed a 98% drop in ribonucleotide pools within 30 hours of starvation.
- Maintained adenylate energy charge and GTP/ATP ratios for 50 hours, dependent on cell-cell contact.
Conclusions:
- Dictyostelium discoideum possesses both de novo and salvage pyrimidine synthesis pathways.
- Developmental transition involves significant nucleotide pool reduction while maintaining energy homeostasis.
- Cell-cell contact is essential for metabolic regulation during Dictyostelium discoideum development.