Identification of purine biosynthesis as an NADH-sensing pathway to mediate energy stress

An enhanced NADH/NAD⁺ ratio, termed reductive stress, is associated with many diseases. However, whether a downstream sensing pathway exists to mediate pathogenic outcomes remains unclear. Here, we generate a soluble pyridine nucleotide transhydrogenase from Escherichia coli (EcSTH), which can elevate the NADH/NAD⁺ ratio and meantime reduce the NADPH/NADP⁺ ratio. Additionally, we fuse EcSTH with previously described LbNOX (a water-forming NADH oxidase from Lactobacillus brevis) to resume the NADH/NAD⁺ ratio. With these tools and by using genome-wide CRISPR/Cas9 library screens and metabolic profiling in mammalian cells, we find that accumulated NADH deregulates PRPS2 (Ribose-phosphate pyrophosphokinase 2)-mediated downstream purine biosynthesis to provoke massive energy consumption, and therefore, the induction of energy stress. Blocking purine biosynthesis prevents NADH accumulation-associated cell death in vitro and tissue injury in vivo. These results underscore the pathophysiological role of deregulated purine biosynthesis in NADH accumulation-associated disorders and demonstrate the utility of EcSTH in manipulating NADH/NAD⁺ and NADPH/NADP⁺.