Fig. 1: Tracing the ¹⁵N labeled glutamine and aspartic acid incorporation in de novo synthesized pyrimidines.

A Schematic describing glutamine (Gln) mediated ¹⁵N label incorporation in pyrimidine de novo biosynthesis intermediates and end product UMP (also see schematic Fig. S1A). Various isotope labeled analytes generated during the course of the assay are: ¹⁵N2 Gln (sidechain amide nitrogen: open red circle, backbone amine nitrogen: solid red circle), ¹⁵N glutamic acid (Glu, backbone amine nitrogen: solid red circle), and ¹⁵N aspartic acid (Asp, backbone amine nitrogen: solid red circle). The pyrimidine nucleotide UMP (N₁ and N₃) with the labeled positions arising from Gln side chain and Asp backbone, respectively, are marked on the representative pyrimidine ring. The enzyme glutamate oxaloacetate transaminase GOT1 (cytosolic) or GOT2 (mitochondria) catalyzes the conversion of oxaloacetic acid (OAA) to Asp, transferring the backbone amine of Glu to Asp. Two alternative models for pyrimidine de novo biosynthesis: B completely diffusive versus C channeled pathway with CAD and UMPS coordinating with DHODH, respectively. C A channeled mechanism-based model for the pyrimidine de novo biosynthesis metabolon, the pyrimidinosome (blue background), with CAD and UMPS that assemble proximal to mitochondria and DHODH, allowing preferential utilization of the mitochondrially generated ¹⁵N Asp. In this model, mitochondrial GOT2 serves as the primary source of ¹⁵N Asp utilized for channeled pyrimidine biosynthesis. Diffusive model predicts homogenous ¹⁵N isotope label incorporation across the various substrates and product, while the channeled pathway predicts isotope label inhomogeneity. Enzyme names are italicized and Asp, Gln mitochondrial transporters are shown in magenta and green, respectively. To test which model applies to pyrimidine synthesis, ¹⁵N2 Gln labeling was carried out for 2 h. The %¹⁵N labeled Asp incorporation in the analytes of interest is shown: D UDP and Asp and E carbamoyl aspartate, dihydroorotate, and orotate. The %¹⁵N labeled Asp incorporation was calculated as =\(\left(M+1.994\right)\div\left[\left(M+.997\right)+\left(M+1.994\right)\right]\). Data from three independent biological replicates are shown, bar heights represent the mean. **p-value < 0.005 for a pairwise comparison using two-way t-test (Additional data in supplementary Fig. S2). A, B, and C prepared using Biorender and Chemdraw structures were used in A https://BioRender.com/x25177d.