PYCR3 (pyrroline-5-carboxylate reductase 3) is a cytosolic oxidoreductase that catalyzes the final step in proline biosynthesis, reducing pyrroline-5-carboxylate (P5C) to L-proline using NAD(P)H as a coenzyme 1. Unlike PYCR1 and PYCR2, PYCR3 is exclusively linked to proline biosynthesis from ornithine rather than glutamate 2. Kinetic studies demonstrate PYCR3 functions through a random ordered bi-bi mechanism and shows activity with both NADPH and NADH 1. In cancer contexts, PYCR3 exhibits significant upregulation in triple-negative breast cancer (TNBC) and correlates with poor prognosis 3. The enzyme localizes to mitochondria in TNBC cells where it regulates mtDNA copy number and mitochondrial respiration, promoting cell proliferation and conferring doxorubicin resistance 3. PYCR3 is stabilized by the deubiquitinase USP9x in non-small cell lung cancer, where its disruption impairs the pentose phosphate pathway and mitochondrial respiration 4. Across multiple cancer types, PYCR3 expression correlates with immune cell infiltration and tumor progression 5. These findings position PYCR3 as both a metabolic regulator and potential therapeutic target in cancer treatment.