Functional profiling reveals a non-enzymatic role of NUDT5 in repressing purine de novo synthesis

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. Here, we investigate the chemical and genetic dependencies caused by mutations in the folate enzyme MTHFD1 and discover a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Through genetic knockout and development of a selective chemical NUDT5 degrader, we uncover an unprecedented scaffolding role rather than NUDT5 enzymatic activity is responsible for this phenotype. We find that NUDT5 interacts with the rate-limiting enzyme of purine de novo synthesis, PPAT, to repress the pathway in response to elevated purine levels. Our findings establish NUDT5 as an important regulator of purine de novo synthesis and elucidate its role in mediating sensitivities to 6-thioguanine in cancer treatment and to adenosine in MTHFD1 deficiency.