Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle

Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5 '-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity. Shin et al. gain structural insight into how pyrimidine synthesis is coupled to cell cycle regulation through the modulation of CAD activity, and identify allostery as a major means to regulate de novo pyrimidine biosynthesis during the mammalian cell cycle.

Automated summary

This study uncovers the mechanism by which human de novo pyrimidine biosynthesis is regulated during the cell cycle. The researchers identify carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as the key enzymes regulated in the cell cycle, and demonstrate that allostery plays a major role in controlling CAD activity. They also find evidence of Animalia-specific loops in the CAD allosteric domain, which are involved in sensing and binding of uridine 5'-triphosphate, an allosteric inhibitor of CAD. Through structural analysis, a critical signal transmission loop is identified in regulating the formation of a substrate channel and controlling CAD activity.