D-serine suppresses one-carbon metabolism by competing with mitochondrial L-serine transport

Abstract L-serine serves as a central metabolic node that integrates glycolytic flux, lipid metabolism, and one-carbon metabolism. In the mature central nervous system, L-serine is actively stereo-converted to D-serine, which functions as a neurotransmitter. However, the role of D-serine in cellular metabolism remains unclear. Here, we show that D-serine competes with mitochondrial L-serine transport, thereby suppressing one-carbon metabolism. Metabolomic analysis revealed that D-serine reduces intracellular glycine and formate levels, indicating inhibition of the initial step of the one-carbon pathway. Molecular dynamics simulations and enzymatic assays revealed that D-serine has low affinity for serine hydroxymethyltransferase 2 (Shmt2), which catalyzes the first step in mitochondrial one-carbon metabolism, and does not directly inhibit its activity. Instead, membrane transport assays demonstrated that D-serine competes with mitochondrial L-serine transport, depleting the substrate of Shmt2. Functionally, under L-serine poor conditions in vitro and ex vivo, D-serine inhibited the proliferation of immature and undifferentiated neural cells including glioblastoma stem cells, which depend highly on one-carbon metabolism. Notably, endogenous D-serine levels were low during early neurodevelopment, but increased with maturation, coinciding with a shift in the transcriptional profiles of serine metabolic enzymes at the cellular level. Given that L-serine supports neurodevelopment and D-serine modulates neurotransmission, this developmental shift in serine enantiomer metabolism appears to align with the functional transitions of the maturing nervous system. Thus, our findings reveal that serine chirality can influence mitochondrial substrate availability and one-carbon flux, offering previously unappreciated insight into the stereoselective regulation of cellular metabolism.